Extended half-life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells

The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli beta-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import/export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the proteasomes. In the absence of antigen, the half-life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with beta-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of beta-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.     

Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework

Single-chain Fv antibody fragments (scFv) represent a convenient antibody format for intracellular expression in eukaryotic or prokaryotic cells. These so-called intrabodies have great potential in functional genomics as a tool to study the function of newly identified proteins in vivo, for example by binding-induced modulation of their activity or by blocking interactions with other proteins. However, the intracellular expression and activity of many single-chain Fvs are limited by their instability and folding efficiency in the reducing intracellular environment, where the highly conserved intrachain disulfide bonds do not form. In the present work, we used an in vivo selection system to isolate novel antigen-binding intrabodies. We screened two intrabody libraries carrying a randomized third hypervariable loop onto the heavy chain of a stable framework, which had been further optimized by random mutagenesis for better behavior in the selection system, and we biophysically characterized the selected variants to interpret the outcome of the selection. Our results show that single-framework intrabody libraries can be directly screened in vivo to rapidly select antigen-specific intrabodies.     

Growth signalobody selects functional intrabodies in the mammalian cytoplasm

A versatile strategy to inhibit protein functions in the cytoplasmic environment is eagerly anticipated for drug discovery. In this study, we demonstrate a novel system to directly select functional intrabodies from a library in the mammalian cytoplasm. In this system, a target homo‐oligomeric antigen is expressed together with a single‐chain Fv (scFv) library that is linked to the cytoplasmic domain of a receptor tyrosine kinase (RTK) in the cytoplasm of murine interleukin‐3 (IL‐3)‐dependent cells. As the tyrosine kinase is activated by dimerization, only scFv‐RTK clones that can bind to the target antigen would be oligomerized and transduce a growth signal under the IL‐3‐deprived condition, which leads to selection of functional intrabodies. To demonstrate this system, we used rabies virus phosphoprotein (RV‐P) that forms dimers in the cytoplasm as a target antigen. As a result, functional intrabodies were selected using our system from a naïve scFv library as well as from a pre‐selected anti‐RV‐P library generated by phage display. This system may be applied for screening intrabodies that can prevent progression of various severe diseases.     

Enhanced intracellular stability of sFv-Fc fusion intrabodies

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.     

Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies

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.     

Antigen-independent selection of stable intracellular single-chain antibodies.

The intracellular expression of single-chain Fv antibody fragments (scFv) in eukaryotic cells has an enormous potential in functional genomics and therapeutics [Marasco (1997) Gene Ther. 4, 11-15; Richardson and Marasco (1995) Trends Biotechnol. 13, 306-310]. However, the application of these so-called intrabodies is currently limited by their unpredictable behavior under the reducing conditions encountered inside eukaryotic cells, which can affect their stability and solubility properties [W?rn et al. (2000) J. Biol. Chem. 275, 2795-2803; Biocca et al. (1995) Bio/Technology 13, 1110-1115]. We present a novel system that enables selection of stable and soluble intrabody frameworks in vivo without the requirement or knowledge of antigens. This system is based on the expression of single-chain antibodies fused to a selectable marker that can control gene expression and cell growth. Our results show that the activity of a selectable marker fused to well characterized scFvs [W?rn et al. (2000) J. Biol. Chem. 275, 2795-2803] correlates with the solubility and stability of the scFv moieties. This method provides a unique tool to identify stable and soluble scFv frameworks, which subsequently serve as acceptor backbones to construct intrabody complementarity determining region libraries by randomization of hypervariable loops.     

Conjugative transfer can be inhibited by blocking relaxase activity within recipient cells with intrabodies

Horizontal transfer of antibiotic resistance genes carried by conjugative plasmids poses a serious health problem. As conjugative relaxases are transported to recipient cells during bacterial conjugation, we investigated whether blocking relaxase activity in the recipient cell might inhibit conjugation. For that purpose, we used an intrabody approach generating a single-chain Fv antibody library against the relaxase TrwC of conjugative plasmid R388. Recombinant single-chain Fv antibodies were engineered for cytoplasmic expression in Escherichia coli cells and either selected in vitro for their specific binding to TrwC, or in vivo by their ability to interfere with conjugation using a high-throughput mating assay. Several intrabody clones were identified showing specific inhibition against R388 conjugation upon cytoplasmic expression in the recipient cell. The epitope recognized by one of these intrabodies was mapped to a region of TrwC containing Tyr-26 and involved in the conjugative DNA-processing termination reaction. These findings demonstrate that the transferred relaxase plays an important role in the recipient cell and open a new approach to identify specific inhibitors of bacterial conjugation.     

Intrabodies based on intracellular capture frameworks that bind the RAS protein with high affinity and impair oncogenic transformation

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.     

Isolation of anti-MISIIR scFv molecules from a phage display library by cell sorter biopanning

While cell surface antigens represent the most common targets for antibody-based cancer therapy, isolation of new antibodies specific for these targets from single-chain Fv phage display libraries has been hindered by limitations associated with traditional selection techniques. Solid phase panning is often associated with conformational changes to the target protein due to its immobilization on plastic tubes that can limit the ability of the isolated scFv to bind to conformational epitopes and solution panning methods require the use of secondary tags that often mask desired sequences and create unintended epitopes. Commonly utilized cell-based panning methods typically yield a panel of single-chain Fv (scFv) molecules that are specific for numerous cell surface antigens, often obscuring the desired clones. Here, we describe a novel cell sorter-based system to isolate single-chain Fv molecules specific for defined antigen targets expressed on stably-transformed mammalian cells. We employed these methods to isolate promising scFv clones that bind specifically to the Müllerian inhibiting substance type II receptor, a cell surface ovarian cancer antigen that has proven to be a difficult target for selection strategies.     

Selective re-routing of prion protein to proteasomes and alteration of its vesicular secretion prevent PrP(Sc) formation

Conversion of the cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is the hallmark of prion diseases, which are fatal and transmissible neurodegenerative disorders. ER-retained anti-prion recombinant single-chain Fv fragments have been proved to be an effective tool for inhibition of PrP(C) trafficking to the cell surface and antagonize PrP(Sc) formation and infectivity. In the present study, we have generated the secreted version of 8H4 intrabody (Sec-8H4) in order to compel PrP(C) outside the cells. The stable expression of the Sec-8H4 intrabodies induces proteasome degradation of endogenous prion protein but does not influence its glycosylation profile and maturation. Moreover, we found a dramatic diverting of PrP(C) traffic from its vesicular secretion and, most importantly, a total inhibition of PrP(Sc) accumulation in NGF-differentiated Sec-8H4 PC12 cells. These results confirm that perturbing the intracellular traffic of endogenous PrP(C) is an effective strategy to inhibit PrP(Sc) accumulation and provide convincing evidences for application of intracellular antibodies in prion diseases.     

Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment

A prerequisite for the enrichment of antibodies screened from phage display libraries is their stable expression on a phage during multiple selection rounds. Thus, if stringent panning procedures are employed, selection is simultaneously driven by antigen affinity, stability and solubility. To take advantage of robust pre-selected scaffolds of such molecules, we grafted single-chain Fv (scFv) antibodies, previously isolated from a human phage display library after multiple rounds of in vitro panning on tumor cells, with the specificity of the clinically established murine monoclonal anti-CD22 antibody RFB4. We show that a panel of grafted scFvs retained the specificity of the murine monoclonal antibody, bound to the target antigen with high affinity (6.4-9.6 nM), and exhibited exceptional biophysical stability with retention of 89-93% of the initial binding activity after 6 days of incubation in human serum at 37 degrees C. Selection of stable human scaffolds with high sequence identity to both the human germline and the rodent frameworks required only a small number of murine residues to be retained within the human frameworks in order to maintain the structural integrity of the antigen binding site. We expect this approach may be applicable for the rapid generation of highly stable humanized antibodies with low immunogenic potential.     

肝癌特异性鼠源及人源化单链抗体基因的构建及大肠杆菌中的表达

为探讨一株肝细胞癌特异性鼠源及其人源化单链抗体基因在大肠杆菌中的可溶性表达策略并比较二者对抗原的结构能力,在三种载体中分别以融合、分泌及胞内表达的方式进行了研究,表达产物均以包涵体形式存在;对复性后的单链抗体以细胞ＥＬＩＳＡ及竞争抑制流式细胞仪法进行检测,表明人源化单链抗体和鼠源单链抗体有相近的抗原结合能力。结论是：大肠杆菌中表达的基因工程单链抗体的可溶性可能主要由自身氨基酸一级序列决定;先前的设     

Development of a human light chain variable domain (V(L)) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display

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.     

Conformational Targeting of Fibrillar Polyglutamine Proteins in Live Cells Escalates Aggregation and Cytotoxicity

Background

Misfolding- and aggregation-prone proteins underlying Parkinson''s, Huntington''s and Machado-Joseph diseases, namely α-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.

Methodology/Principal Findings

Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of α-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.

Conclusions/Significance

These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases.     

Expression of a functional single-chain antibody against GA24/19 in transgenic tobacco.

An anti-gibberellin A24/19 single-chain Fv gene was constructed from gamma and kappa genes cloned from a hybridoma cell line producing monoclonal antibody against gibberellin A24/19, biosynthetic precursors of gibberellin A4/1 which are biologically active per se. The single-chain Fv gene was introduced into tobacco plants after the binding activity of the single-chain Fv expressed in Escherichia coli was confirmed. When the single-chain Fv expression is targeted to endoplasmic reticulum, the plants could accumulate the single-chain Fv protein with the antigen binding activity up to 3.6% of the total soluble protein. On the other hand, when the expression is targeted to cytosol, accumulation of the single-chain Fv protein was not detected at all. The dwarf phenotype of the transgenic plants expressing the single-chain Fv protein, together with the preliminary analytical data indicating a decreased level of gibberellin A1 in the dwarf transgenics, suggested that the single-chain Fv decreased the concentration of bioactive gibberellins by trapping and inhibiting the metabolism of gibberellin A24 and/or A19 to gibberellin A4 and/or A1.     

Engineering stable cytoplasmic intrabodies with designed specificity

Many attempts have been made to develop antibody fragments that can be expressed in the cytoplasm ("intrabodies") in a stable and functional form. The recombinant antibody fragment scFv(F8) is characterised by peculiarly high in vitro stability and functional folding in both prokaryotic and eukaryotic cytoplasm. To dissect the relative contribution of different scFv(F8) regions to cytoplasmic stability and specificity we designed and constructed five chimeric molecules (scFv-P1 to P5) in which several groups of residues important for antigen binding in the poorly stable anti-hen egg lysozyme (HEL) scFv(D1.3) were progressively grafted onto the scFv(F8) scaffold. All five chimeric scFvs were expressed in a soluble form in the periplasm and cytoplasm of Escherichia coli. All the periplasmic oxidised forms and the scFv(P3) extracted from the cytoplasm in reducing conditions had HEL binding affinities essentially identical (K(d)=15nM) to that of the cognate scFv(D1.3) fragment (K(d)=16nM). The successful grafting of the antigen binding properties of D1.3 onto the scFv(F8) opens the road to the exploitation of this molecule as a scaffold for the reshaping of intrabodies with desired specificities to be targeted to the cytoplasm.     

Improved yield and stability of L49-sFv-beta-lactamase, a single-chain antibody fusion protein for anticancer prodrug activation, by protein engineering

The L49 single-chain Fv fused to beta-lactamase (L49-sFv-bL) combined with the prodrug C-Mel is an effective anticancer agent against tumor cells expressing the p97 antigen. However, large-scale production of L49-sFv-bL from refolded E. coli inclusion bodies has been problematic due to inefficient refolding and instability of the fusion protein. Sequence analysis of the L49-sFv framework regions revealed three residues in the framework regions at positions L2, H82B, and H91, which are not conserved for their position, occurring in <1% of sequences in Fv sequence databases. One further unusual residue, found in <3% of variable sequences, was observed at position H39. Each unusual residue was mutated to a conserved residue for its position and tested for refolding yield from inclusion bodies following expression in E. coli. The three V(H) single mutants showed improvement in the yield of active protein and were combined to form double and triple mutants resulting in a 7-8-fold increased yield compared to the parental protein. In an attempt to further improve yield, the orientation of the triple mutant was reversed to create a bL-L49-sFv fusion protein resulting in a 3-fold increase in expressed inclusion body protein and producing a 20-fold increase in the yield of purified protein compared to the parental protein. The triple mutants in both orientations displayed increased stability in murine plasma and binding affinity was not affected by the introduced mutations. Both triple mutants also displayed potent in vitro cytotoxicity and in vivo antitumor activity against p97 expressing melanoma cells and tumor xenografts, respectively. These results show that a rational protein-engineering approach improved the yield, stability, and refolding characteristics of L49-sFv-bL while maintaining binding affinity and therapeutic efficacy.     

Intradiabodies,bispecific, tetravalent antibodies for the simultaneous functional knockout of two cell surface receptors

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.     

Intracellular targeting and functional analysis of single-chain Fv fragments in mammalian cells

In the past decade, intracellular antibodies have proven to be a useful tool in obtaining the phenotypic knock-out of selected gene function in different animal and plant systems. This strategy is based on the ectopic expression of recombinant forms of antibodies targeted towards different intracellular compartments, exploiting specific targeting signals to confer the new intracellular location. The functional basis of this technology is closely linked to the ability of intracellular antibodies to interact with their target antigens in vivo. This interaction allows either a direct neutralising effect or the dislodgement of the target protein from its normal intracellular location and, by this mechanism, the inactivation of its function. By using this approach, the function of several antigens has been inhibited in the cytoplasm, the nucleus, and the secretory compartments. In this article, we shall describe all the steps required for expressing single-chain Fv fragments in different subcellular compartments of mammalian cells and their subsequent use in knock-out experiments, starting from a cloned single-chain Fv fragment. This will include the analysis of the solubility properties of the new scFv fragment in transfected mammalian cells, the intracellular distribution of the antigen-antibody complex, and the resulting phenotype.