A focused antibody library for selecting scFvs expressed at high levels in the cytoplasm

Background  

Intrabodies are defined as antibody molecules which are ectopically expressed inside the cell. Such intrabodies can be used to visualize or inhibit the targeted antigen in living cells. However, most antibody fragments cannot be used as intrabodies because they do not fold under the reducing conditions of the cell cytosol and nucleus.     

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.     

Specific in vivo knockdown of protein function by intrabodies

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.     

De novo production of diverse intracellular antibody libraries

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.     

Fusion to a highly charged proteasomal retargeting sequence increases soluble cytoplasmic expression and efficacy of diverse anti-synuclein intrabodies

Intrabodies can be powerful reagents to effect modulation of aberrant intracellular proteins that underlie a range of diseases. However, their cytoplasmic solubility can be limiting. We previously reported that overall charge and hydrophilicity can be combined to provide initial estimates of intracellular solubility, and that charge engineering via fusion can alter solubility properties experimentally. Additional studies showed that fusion of a proteasome-targeting PEST motif to the anti-huntingtin intrabody scFv-C4 can degrade mutant huntingtin proteins by directing them to the proteasome, while also increasing the negative charge. We now validate the generality of this approach with intrabodies against α-synuclein (α-syn), an important target in Parkinson disease. In this study, fusion of the PEST sequence to a set of four diverse, poorly soluble anti-α-syn intrabodies (D5E, 10H, D10 scFv, VH14 nanobody) significantly increased steady-state soluble intrabody protein levels in all cases, despite fusion with the PEST proteasomal-targeting signal. Furthermore, adding this PEST motif to the least soluble construct, VH14, significantly enhanced degradation of the target protein, α-syn~GFP. The intrabody-PEST fusion approach thus has dual advantages of potentially solubilizing intrabodies and enhancing their functionality in parallel. Empirical testing of intrabody-PEST fusions is recommended for enhancement of intrabody solubility from diverse sources.     

Fusion to a highly charged proteasomal retargeting sequence increases soluble cytoplasmic expression and efficacy of diverse anti-synuclein intrabodies

Intrabodies can be powerful reagents to effect modulation of aberrant intracellular proteins that underlie a range of diseases. However, their cytoplasmic solubility can be limiting. We previously reported that overall charge and hydrophilicity can be combined to provide initial estimates of intracellular solubility, and that charge engineering via fusion can alter solubility properties experimentally. Additional studies showed that fusion of a proteasome-targeting PEST motif to the anti-huntingtin intrabody scFv-C4 can degrade mutant huntingtin proteins by directing them to the proteasome, while also increasing the negative charge. We now validate the generality of this approach with intrabodies against α-synuclein (α-syn), an important target in Parkinson disease. In this study, fusion of the PEST sequence to a set of four diverse, poorly soluble anti-α-syn intrabodies (D5E, 10H, D10 scFv, VH14 nanobody) significantly increased steady-state soluble intrabody protein levels in all cases, despite fusion with the PEST proteasomal-targeting signal. Furthermore, adding this PEST motif to the least soluble construct, VH14, significantly enhanced degradation of the target protein, α-syn~GFP. The intrabody-PEST fusion approach thus has dual advantages of potentially solubilizing intrabodies and enhancing their functionality in parallel. Empirical testing of intrabody-PEST fusions is recommended for enhancement of intrabody solubility from diverse sources.     

beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide

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.     

Generation of functional scFv intrabodies for triggering anti-tumor immunity

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.     

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.     

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.     

Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules

The use of antibodies in medicine and research depends on their specificity and affinity in the recogniton and binding of individual molecules. However, these applications are limited to the extracellular targets. Advances in antibody engineering has allowed the manipulation of the antibody segments containing the antigen-binding regions and generation of small fragments that can be stably expressed in cells. These entities are called intracellular antibodies or intrabodies and have being successfully applied, mainly in the scFv format, to inhibit the function of intracellular target proteins in specific cellular compartments. As new techniques to select and isolate intrabody fragments have been developed, intrabodies are beginning to be used to interfere with the function of a greater number of relevant disease targets. Just as monoclonal antibodies are opening a new era in human therapeutics, intrabodies promise a new prospective for antibody tools for therapy and research. Their varied mode of action gives intrabodies great potential in different approaches in the treatment of human diseases, as well as in the area of functional genomics for characterisation of novel gene products and subsequent validation as potential drug targets. While techniques for identifying functional intrabodies have improved, there are still many significant problems to be overcome before intrabodies can actually be used in treatment of diseases such as cancer, AIDS or neuro-degenerative disorders.     

Generation of an intrabody‐based reagent suitable for imaging endogenous proliferating cell nuclear antigen in living cancer cells

Intrabodies, when expressed in cells after genetic fusion to fluorescent proteins, are powerful tools to study endogenous protein dynamics inside cells. However, it remains challenging to determine the conditions for specific imaging and precise labelling of the target antigen with such intracellularly expressed antibody fragments. Here, we show that single‐chain Fv (scFv) antibody fragments can be generated that specifically recognize proliferating cell nuclear antigen (PCNA) when expressed in living cancer cells. After selection by phage display, the anti‐PCNA scFvs were screened in vitro after being tagged with dimeric glutathione‐S‐transferase. Anti‐PCNA scFvs of increased avidity were further engineered by mutagenesis with sodium bisulfite and error‐prone PCR, such that they were almost equivalent to conventional antibodies in in vitro assays. These intrabodies were then rendered bifunctional by fusion to a C‐terminal fragment of p21 protein and could thereby readily detect PCNA bound to chromatin in cells. Finally, by linking these optimized peptide‐conjugated scFvs to an enhanced green fluorescent protein, fluorescent intrabody‐based reagents were obtained that allowed the fate of PCNA in living cells to be examined. The approach described may be applicable to other scFvs that can be solubly expressed in cells, and it provides a unique means to recognize endogenous proteins in living cells with high accuracy. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Cloning of apoB intrabodies: specific knockdown of apoB in HepG2 cells

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.     

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.     

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

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.     

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.     

Functional knock down of VCAM1 in mice mediated by endoplasmatic reticulum retained intrabodies

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.     

Intracellular antibodies and challenges facing their use as therapeutic agents

A key feature of antibodies is their ability to bind antigens with high specificity and affinity. This has led to the concept of intracellular antibodies (intrabodies), designed to mimic antibody-antigen binding, but inside cells. Antibody fragments comprising the antigen-binding variable domains are convenient formats for intrabodies, potentially allowing for intracellular functionality. Intrabodies are promising tools, capable of interfering with a wide range of molecular targets in various intracellular compartments. However, many significant challenges remain to be overcome before intrabodies can be useful therapeutic agents. Although major progress has been made in the design and selection of intrabodies, new developments and advances are needed to allow their efficient delivery and expression for treatment of human diseases.     

KDEL-tagged anti-prion intrabodies impair PrP lysosomal degradation and inhibit scrapie infectivity

Transmissible spongiform encephalopathy or prion diseases are fatal neurodegenerative disorders characterized by the conversion of the cellular prion protein (PrPC) into the infectious scrapie isoform (PrPSc). We have recently demonstrated that anti-prion intrabodies targeted to the lumen of the endoplasmic reticulum provide a simple and effective means to inhibit the transport of PrPC to the cell surface. Here, we report that they completely block the traffic of mature full-length PrPC molecules, impair prion lysosomal degradation, and interfere with the early phase of scrapie formation. Since anti-prion intrabodies efficiently block PrPSc accumulation in vitro, we investigated whether they could also antagonize scrapie infectivity in vivo. We found that mice intracerebrally injected with KDEL-8H4-NGF-differentiated PC12 cells infected with scrapie neither develop scrapie clinical signs nor brain damage. Furthermore, no protease-resistant PrPSc is detectable in brains of inoculated animals. These results indicate that anti-prion intrabody strategy may be effective against prion infection.