Intrabody Expression Research Articles

Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy

Xenophagy, a type of selective autophagy, is a bactericidal membrane trafficking that targets cytosolic bacterial pathogens, but the membrane homeostatic system to cope with bacterial infection in xenophagy is not known. Here, we show that the endosomal sorting complexes required for transport (ESCRT) machinery is needed to maintain homeostasis of xenophagolysosomes damaged by a bacterial toxin, which is regulated through the TOM1L2–Rab41 pathway that recruits AAA-ATPase VPS4. We screened Rab GTPases and identified Rab41 as critical for maintaining the acidification of xenophagolysosomes. Confocal microscopy revealed that ESCRT components were recruited to the entire xenophagolysosome, and this recruitment was inhibited by intrabody expression against bacterial cytolysin, indicating that ESCRT targets xenophagolysosomes in response to a bacterial toxin. Rab41 translocates to damaged autophagic membranes via adaptor protein TOM1L2 and recruits VPS4 to complete ESCRT-mediated membrane repair in a unique GTPase-independent manner. Finally, we demonstrate that the TOM1L2–Rab41 pathway-mediated ESCRT is critical for the efficient clearance of bacteria through xenophagy.

Selective targeting of intracellular, misfolded, pathogenic TDP‐43 with rationally designed intrabodies

AbstractBackgroundCytoplasmically mislocalized aggregates of TDP‐43 have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and limbic‐predominant age‐related TDP‐43 encephalopathy (LATE) through direct toxicity, loss of function of normal TDP‐43, induction of misfolding of other neuronal proteins, and prion‐like, cell‐to‐cell propagation of disease. We generated monoclonal antibodies (mAbs) selective for the misfolded form of TDP‐43 and derived single chain intrabody constructs to allow for intracellular targeting of pathogenic TDP‐43 without interfering with physiological forms of TDP‐43 important for normal cell function.MethodMonoclonal antibodies (mAbs) were generated by immunization with an unfolded N‐terminal domain (NTD) linear epitope predicted to become exposed in cytoplasmically mislocalized aggregates of TDP‐43 but otherwise buried in natively folded TDP‐43. The selectivity of mAbs for pathogenic, aggregated TDP‐43 was assessed by immunocytochemistry (ICC) of HEK293FT cells transfected with mutant TDP‐43 (DNLS‐TDP‐43) and by immunohistochemistry (IHC) on patient samples. Single chain (scFv) intrabody constructs were derived from selective antibodies and HEK293FT cells were co‐transfected with plasmids encoding intrabodies and misfolding DNLS‐TDP‐43. The selective interaction of expressed intrabodies with cytoplasmic DNLS‐TDP‐43 aggregates was assessed by ICC, and clearance of the aggregates was evaluated by western blot analysis of cell lysates.ResultICC showed mAb reactivity with cytoplasmic aggregates of transfected DNLS‐TDP‐43 but not endogenous wild‐type nuclear TDP‐43. IHC of ALS and FTLD CNS sections confirmed immunoreactivity specifically with pathogenic TDP‐43 in patient samples. Similarly, co‐transfection of DNLS‐TDP‐43 and scFv intrabody constructs showed intracellular interaction of intrabodies with misfolded cytoplasmic aggregates of TDP‐43 but not with normal, nuclear TDP‐43. In agreement with a lack of interference with normal TDP‐43, the robust expression of intrabodies did not affect cell viability. Intracellular clearance of the aggregates was observed in western blots of the cell lysates and was enhanced by the inclusion of a lysosomal targeting signal in the intrabody constructs.ConclusionImmunization with an NTD epitope of misfolded TDP‐43 gave rise to mAbs selective for pathogenic vs physiologically important forms of TDP‐43. Intrabody constructs derived from these mAbs were able to promote clearance of intracellular pathogenic TDP‐43 without toxicity to the cells.

Nanobody click chemistry for convenient site-specific fluorescent labelling, single step immunocytochemistry and delivery into living cells by photoporation and live cell imaging

While conventional antibodies have been an instrument of choice in immunocytochemistry for some time, their small counterparts known as nanobodies have been much less frequently used for this purpose. In this study we took advantage of the availability of nanobody cDNAs to site-specifically introduce a non-standard amino acid carrying an azide/alkyne moiety, allowing subsequent Cu(I)-catalyzed Azide-Alkyne Click Chemistry (CuAAC). This generated a fluorescently labelled nanobody that can be used in single step immunocytochemistry as compared to conventional two step immunocytochemistry. Two strategies were explored to label nanobodies with Alexa Fluor 488. The first involved enzymatic addition of an alkyne-containing peptide to nanobodies using sortase A, while the second consisted of incorporating para-azido phenylalanine at the nanobody C-terminus. Through these approaches, the fluorophore was covalently and site-specifically attached. It was demonstrated that cortactin and β-catenin, cytoskeletal and adherens junction proteins respectively, can be imaged in cells in this manner through single step immunocytochemistry. However, fixation and permeabilization of cells can alter native protein structure and form a dense cross-linked protein network, encumbering antibody binding. It was shown that photoporation prior to fixation not only allowed delivery of nanobodies into living cells, but also facilitated β-catenin nanobody Nb86 imaging of its target, which was not possible in fixed cells. Pharmacological inhibitors are lacking for many non-enzymatic proteins, and it is therefore expected that new biological information will be obtained through photoporation of fluorescent nanobodies, which allows the study of short term effects, independent of gene-dependent (intrabody) expression.

Single-domain intrabodies against hepatitis C virus core inhibit viral propagation and core-induced NFκB activation.

Hepatitis C virus (HCV) core plays a key role in viral particle formation and is involved in viral pathogenesis. Here, constructs for single-domain intrabodies consisting of variable regions derived from mouse mAbs against HCV core were established. Expressed single-domain intrabodies were shown to bind to HCV core, and inhibit the growth of cell culture-produced HCV derived from JFH-1 (genotype 2a) and a TH (genotype 1b)/JFH-1 chimera. Adenovirus vectors expressing intrabodies were also capable of reducing HCV propagation. Intrabody expression did not affect viral entry or genome replication of single-round infectious trans-complemented HCV particles. However, intrabody expression reduced intracellular and extracellular infectious titres in CD81-defective Huh7-25 cells transfected with the HCV genome, suggesting that these intrabodies impair HCV assembly. Furthermore, intrabody expression suppressed HCV core-induced NFκB promoter activity. These intrabodies may therefore serve as tools for elucidating the role of core in HCV pathogenesis.

Intrabody-mediated diverting of HP1β to the cytoplasm induces co-aggregation of H3–H4 histones and lamin-B receptor

Diverting a protein from its intracellular location is a unique property of intrabodies. To interfere with the intracellular traffic of heterochromatin protein 1β (HP1β) in living cells, we have generated a cytoplasmic targeted anti-HP1β intrabody, specifically directed against the C-terminal portion of the molecule. HP1β is a conserved component of mouse and human constitutive heterochromatin involved in diverse nuclear functions including gene silencing, DNA repair and nuclear membrane assembly. We found that the anti-HP1β intrabody sequesters HP1β into cytoplasmic aggregates, inhibiting its traffic to the nucleus. Lamin B receptor (LBR) and a subset of core histones (H3/H4) are also specifically co-sequestered in the cytoplasm of anti-HP1β intrabody-expressing cells. Methylated histone H3 at K9 (Me9H3), a marker of constitutive heterochromatin, is not affected by the anti-HP1β intrabody expression. Hyper-acetylating conditions completely dislodge H3 from HP1β:LBR containing aggregates. The expression of anti-HP1β scFv fragments induces apoptosis, associated with an alteration of nuclear morphology. Both these phenotypes are specifically rescued either by overexpression of recombinant full length HP1β or by HP1β mutant containing the chromoshadow domain, but not by recombinant LBR protein. The HP1β-chromodomain mutant, on the other hand, does not rescue the phenotypes, but does compete with LBR for binding to HP1β. These findings provide new insights into the mode of action of cytoplasmic-targeted intrabodies and the interaction between HP1β and its binding partners involved in peripheral heterochromatin organisation.

Functional inhibition of transitory proteins by intrabody-mediated retention in the endoplasmatic reticulum

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.

Selection for intrabody solubility in mammalian cells using GFP fusions

Single-chain antibody fragments (scFv) expressed in the cytoplasm of mammalian cells, also called intrabodies, have many applications in functional proteomics. These applications are, however, limited by the aggregation-prone behaviour of many intrabodies. We show here that two scFv with highly homologous sequences and comparable soluble expression levels in Escherichia coli cytoplasm have different behaviours in mammalian cells. When over-expressed, one of the scFv aggregates in the cytoplasm whereas the second one is soluble and active. When expressed at low levels, using a retroviral vector, as a fusion with the green fluorescent protein (GFP) the former does not form aggregates and is degraded, resulting in weakly fluorescent cells, whereas the latter is expressed as a soluble protein, resulting in strongly fluorescent cells. These data suggest that the GFP signal can be used to evaluate the soluble expression of intrabodies in mammalian cells. When applied to a subset of an E.coli-optimised intrabody library, we showed that the population of GFP+ cells contains indeed soluble mammalian intrabodies. Altogether, our data demonstrate that the requirements for soluble intrabody expression are different in E.coli and mammalian cells, and that intrabody libraries can be directly optimised in human cells using a simple GFP-based assay.

Inhibition of protease-inhibitor-resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small-molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing.

Physico-chemical determinants of soluble intrabody expression in mammalian cell cytoplasm.

Soluble antibody fragments are desirable not only as potential therapeutic and diagnostic agents for extracellular targets but also as 'intrabodies' for functional genomics, proteomics and gene therapy inside cells. However, antibody fragments are notoriously aggregation-prone when expressed intracellularly, due in part to unfavorable redox potential and macromolecular crowding in cell cytoplasm. Only a small proportion of intrabodies are soluble in cytoplasm and little is known about the sequence determinants that confer such stability. By comparing the cytoplasmic expression of several related human single-chain variable fragments and camelid V(HH)s in mammalian cells, we report that intrabody solubility is highly influenced by CDR content and is improved by an overall negative charge at cytoplasmic pH and reduced hydrophilicity. We hypothesize that ionic repulsion and weak hydrophobic interactions compensate, to different extents, for impaired disulfide bond formation in cytoplasm, thereby decreasing the risk for intrabody aggregation. As proof of principle, we demonstrate that the soluble expression of an aggregation-prone positively charged intrabody is modestly enhanced via cis or trans acidification using highly charged peptide tags (3XFLAG tag, SV40 NLS). These findings suggest that simple sequence analysis and electrostatic manipulation may aid in predicting and engineering solubility-enhanced intrabodies from antibody libraries for intracellular use.

Intracellular Expression of a Single Domain Antibody Reduces Cytotoxicity of 15-Acetyldeoxynivalenol in Yeast

15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V(H)H) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V(H)H immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V(H)H within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 mug.ml(-1). We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 mug.ml(-1). The proof of concept established within this eukaryotic system suggests that in planta V(H)H expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops.

Conditional Inactivation of HIF-1 Using Intrabodies

Hypoxia is a hallmark of solid cancers and triggers the transcription of genes responsible for cell survival. The transcription factor Hypoxia-Inducible Factor 1 (HIF-1) is a key regulator in this response and frequently activated in human cancer. HIF-1 activation is associated with tumor aggressiveness and poor clinical outcome and, therefore, may provide an attractive therapeutic target. Here we provide a novel approach for HIF-1 targeted therapy using single-domain llama antibodies directed against the HIF-1α oxygen dependent degradation domain which encompass the N-terminal transactivation domain. Conditional expression of HIF intrabodies in mammalian cells interfered with binding to pVHL and inhibited hypoxia induced activation of endogenous target genes. Inducible intrabody targeting is a highly specific strategy for temporal protein inactivation and may have applications for disease treatment.

Conditional inactivation of HIF-1 using intrabodies.

Hypoxia is a hallmark of solid cancers and triggers the transcription of genes responsible for cell survival. The transcription factor Hypoxia-Inducible Factor 1 (HIF-1) is a key regulator in this response and frequently activated in human cancer. HIF-1 activation is associated with tumor aggressiveness and poor clinical outcome and, therefore, may provide an attractive therapeutic target. Here we provide a novel approach for HIF-1 targeted therapy using single-domain llama antibodies directed against the HIF-1α oxygen dependent degradation domain which encompass the N-terminal transactivation domain. Conditional expression of HIF intrabodies in mammalian cells interfered with binding to pVHL and inhibited hypoxia induced activation of endogenous target genes. Inducible intrabody targeting is a highly specific strategy for temporal protein inactivation and may have applications for disease treatment.

Transfection with Anti-p65 Intrabody Suppresses Invasion and Angiogenesis in Glioma Cells by Blocking Nuclear Factor-κB Transcriptional Activity

The strategy of intracellular antibodies to neutralize the function of target proteins has been widely developed for cancer research. This study used an intrabody against p65 subunit to prevent nuclear factor kappaB (NF-kappaB) transcriptional activity in glioma cells and to inhibit the expression of its target genes involved in the invasion and angiogenesis of human gliomas. A single-chain fragment of antibody variable region (scFv) against p65 was prepared using phage display technique. We then prepared an anti-p65 intrabody construct (pFv/nu) by cloning the scFv-encoding sequence into the mammalian nuclear-targeting vector, pCMV/myc/nuc. p65 expression in human glioma cells (U251 and] U87) transfected with pFv/nu was significantly decreased. We showed that NF-kappaB nuclear translocation and its DNA binding activity were blocked via intrabody transfection in electrophoretic mobility shift assays and the inhibition of NF-kappaB activity in nucleus resulted in the decreasing expression and bioactivity of matrix metalloproteinase-9, urokinase-type plasminogen activator receptor, urokinase-type plasminogen activator, and vascular endothelial growth factor. The intrabody transfected glioma cells showed a markedly lower level of invasion in Matrigel invasion assay. The capillary-like structure formation of endothelial cells was also repressed by coculture with the intrabody transfected glioma cells or exposure to their conditional medium. Intrabody transfection neither induced apoptosis nor altered cell proliferation in U251 and U87 cells as compared with the control vector pCMV/nu. After the injection of pFv/nu-transfected glioma cells, preestablished tumors were almost completely regressed when compared with mock, pCMV/nu, and pGFP/nu. Blocking NF-kappaB activity via the nuclear intrabody expression might be a potential approach for cancer therapy.

T-cell protection and enrichment through lentiviral CCR5 intrabody gene delivery

CCR5 is the chemokine co-receptor for R5-tropic human immunodeficiency virus type 1 (HIV-1) isolates most often associated with primary infection. We have developed an HIV-1 self-inactivating vector, CAD-R5, containing a CCR5 single-chain antibody (intrabody) gene, which when expressed in T-cell lines and primary CD4+ T cells disrupts CCR5 cell surface expression and provides protection from R5-tropic isolate exposure. Furthermore, CAD-R5 intrabody expression in primary CD4+ T cells supports significant growth and enrichment over time during HIV-1-pulsed dendritic cell-T-cell interactions. These results indicate that CCR5 intrabody-expressing CD4+ T cells are refractory against this highly efficient primary route of infection. CD34+ cells transduced with the CAD-R5 vector gave rise to CD4+ and CD8+ thymocytes in non-obese diabetic (NOD)/ severely combined-immunodeficient (SCID)-human thymus/liver (hu thy/liv) mice, suggesting that CCR5 intrabody expression can be maintained throughout differentiation without obvious cellular effects. CD4+ T cells isolated from NOD/SCID-hu thy/liv mice were resistant to R5-tropic HIV-1 challenge demonstrating the maintenance of protection. Our findings demonstrate delivery of anti-HIV-1 activity through CCR5 intrabodies in primary CD4+ T cells and CD34+ cell-derived T-cell progeny. Thus, gene delivery strategies that provide a selective survival and growth advantage for T effector cells may provide a therapeutic benefit for HIV-1-infected individuals who have failed conventional therapies.

247. Devising Aβ-Specific Passive Immunization Strategies for Alzheimer's Disease Using rAAV Vector-Mediated scFv Antibody Delivery

Amyloid beta (A|[beta]|) has been identified as a key pathological component in Alzheimer's disease (AD). The accumulation of intraneuronal and extracellular A|[beta]| coincides with the cognitive deficits associated with disease progression and is believed to underlie these deficits. Significant in vitro and human pathological data suggest that intraneuronal accumulation of A|[beta]| peptides plays an early triggering role in the cascade ultimately leading to AD-related neurodegeneration. Recently, a triple transgenic mouse model of AD (3xTg-AD) that develops both amyloid and tau pathology has been created, which to date is the most disease-relevant model system depicting what occurs in human AD. We and others have observed significant accumulation of intraneuronal A|[beta]| in these mice at 3 months of age, a time point prior to the onset of demonstrable amyloid plaque and tau pathologies, but similar temporally to when synaptic dysfunction is electrophysiologically detectable. A|[beta]|-specific intracellular antibodies (intrabodies) with differing intracellular trafficking characteristics were engineered from a previously obtained secretion-competent single-chain antibody (scFv) to interact with A|[beta]| intracellularly and promote its degradation. Cell lines have been created that harbor a doxycycline-regulated transcription unit expressing the Swedish mutant form of human amyloid precursor protein and in essence, intracellular human A|[beta]| peptides. Intrabody constructs are being assessed in these lines for their abilities to modulate A|[beta]| intracellular localization and half-life. Recombinant adeno-associated virus (rAAV) vectors expressing the parental scFv's and the engineered intrabodies are being administered to 3xTg-AD mice prior to the initial appearance of the intraneuronal A|[beta]| signature. In pilot in vivo studies, soluble and membrane-bound forms of the A|[beta]|-specific scFv were intra-cranially administered to the CA1 region of the hippocampus in 2-month-old 3xTg-AD mice. Mice were sacrificed at 8 months post-injection, and brains were processed for immunohistochemical analyses. Results collected to date strongly suggest that this passive immunotherapeutic modality leads to clearance of A|[beta]| in hippocampal regions. Future work involving rAAV-mediated intrabody expression will provide major insights into the involvement of intraneuronal A|[beta]| accumulation in the temporal and spatial progression of early AD pathogenic events and potentially leading to the development of new anti-A|[beta]| therapeutics. Supported by NIH R01AG020204 to HJF and NIH R01AG023593 to WJB.

Intrabodies targeting the Kaposi sarcoma–associated herpesvirus latency antigen inhibit viral persistence in lymphoma cells

Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen-1 (LANA1) is essential for the maintenance and segregation of viral episomes in KSHV latently infected B cells. We report development of intracellular, rabbit-derived antibodies generated by phage display technology, which bind to N-terminal LANA1 epitopes and neutralize the chromosome-binding activity of LANA1. Although these cloned single-chain variable fragments (scFvs) show relatively low binding affinities for the LANA1 viral antigen in in vitro assays, they nonetheless outcompete KSHV-seropositive human sera for LANA1 epitope binding. In heterologous cells, intracellular intrabody expression inhibits LANA1-dependent plasmid maintenance of both an artificial plasmid containing KSHV LANA1 binding sequences and a bacterial artificial chromosome containing the entire KSHV genome. In KSHV naturally infected primary effusion lymphoma cells, intracellular intrabody expression causes a reduction or loss of the typical LANA1 punctate, nuclear pattern. This morphologically apparent LANA1 dispersion correlates to loss of viral episome by molecular analysis. These data suggest a novel approach to antiherpes viral therapy and confirm LANA1 is critical target for neutralization of KSHV viral latency.

Intrabodies against the EVH1 domain of Wiskott–Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells

Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single-chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (V(H)) leader signal sequence at the N-terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott-Aldrich syndrome protein (WASP). WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin (IL)-2 synthesis in the T-cell receptor (TCR) signaling pathway. It has been suggested that an EVH1 domain in the N-terminal region of WASP may participate in IL-2 synthesis. In transgenic mice expressing anti-EVH1 scFvs derived from hybridoma cells producing WASP-EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti-EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL-2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T-cell defects in WASP transgenic mice overexpressing the EVH1 domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 domain function that regulates IL-2 synthesis signaling without affecting the overall domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.

A universal strategy for stable intracellular antibodies

The expression of intracellular antibodies (intrabodies) in mammalian cells has provided a powerful tool to manipulate microbial and cellular signalling pathways in a highly precise manner. However, several technical hurdles have thus far restricted their more widespread use. In particular, single-chain antibodies (scFvs) have been reported to fold poorly in the reducing environment of the cytoplasm and as such there has been a reluctance to use scFv-phage libraries as a source of intrabodies unless a preselection step was applied to identify these rare scFvs that could fold properly in the absence of disulfide bonds. Recently, we reported that scFvs can be efficiently expressed within the cytoplasm of bacteria when fused at the C-terminus of the Escherichia coli maltose-binding protein (MBP). Here, we demonstrate that such MBP–scFvs are similarly stabilized when expressed in the mammalian cell cytoplasm as well as other compartments. This was demonstrated by comparing MBP–scFv fusions to the corresponding unfused scFvs that activate a defective β-galactosidase enzyme, others that neutralize the wild-type β-galactosidase enzyme, and an antibody that blocks the epidermal growth factor receptor. In all cases, the MBP–scFvs significantly outperformed their unfused counterparts. Our results suggest that fusion of scFvs to MBP, and possibly to other “chaperones in the context of a fusion protein,” may provide a universal approach for efficient expression of intrabodies in the mammalian cell cytoplasm. This strategy should allow investigators to bypass much of the in vitro scFv characterization that is often not predictive of in vivo intrabody function and provide a more efficient use of large native and synthetic scFv-phage libraries already in existence to identify intrabodies that will be active in vivo.

973. Targeting of CCR5 through Intrabody Mediated HIV-1 Vector Gene Delivery Is Sufficient for Blocking HIV-1-Infection and Confers a T-Cell Survival and Growth Advantage

Top of pageAbstract With the emergence of HAART resistant HIV-1s, there is a need for additional therapies to preserve T-effector cells and reduce viral burden. Moreover, since it may be impossible to protect all susceptible cells in an individual through gene delivery, it is therefore necessary to develop and verify that genetic means of protection against HIV-1 provides a cellular survival and growth advantage. To this purpose we have developed single chain antibodies to CCR5 which targets protein. Efficacy of HIV-1 protection and enrichment was assessed by free viral and HIV-1 loaded dendritic cell challenge. HIV-1 vector delivery of CCR5-intrabody (termed the CAD-R5 vector), coupled to a KDEL endoplasmic retention sequence, to primary CD4 T-cells efficiently disrupted CCR5 cell surface expression 4-fold as determined by mean fluorescence intensity (MFI) as compared to a control vector without the intrabody, 8 and 34 MFI, respectfully. CAD-R5 transduced primary CD4 T-cells were resistant to robust, free, R5-tropic SF162 challenges as shown by 50 to 60-fold reductions in HIV-1 p24 concentrations. Moreover, CCR5-intrabody gene expressing CD4 T-cells, as compared to cells with a control vector, demonstrated a selective advantage and enriched from 2-10-fold in dendritic cell cultures. Similar levels of HIV-1 protection were seen in CD34 cell derived macrophages; 3-weeks after challenge with SF162, macrophages expressing CCR5-intrabody generated 42-, 57-, and 52-fold less p24 as compared to parental and control vector containing macrophages. Thus, CCR5 intrabody expression drastically decreased viral loads in CD4 T-cells and macrophages. To assess hematopoietic developmental alterations from CCR5-intrabody expression, CD34 cells were transduced and colony forming (CFU) and macrophage assays were performed. No differences were seen in numbers/types of macrophages or CFUs produced when comparing parental, control vector, and intrabody expressing cells (n=3). To assess in vivo correlates of hematopoietic function and intrabody-mediated protection, CAD-R5 and control vector transduced, fetal liver derived CD34 cells were engrafted in human thymi implants in SCID-hu thy/liv mice (n = 2, 2 groups), 6-weeks later the thymi displayed an average of 25% reporter gene expressing T-cells, similar to controls. Development was not altered as compared to controls as determined by CD4/CD8 profile. Furthermore, CCR5-intrabody expressing thymocytes were also highly resistant to ex vivo R5-tropic HIV-1 challenge. These results validate the efficacy of HIV-1 delivered CCR5-intrabody mediated protection from HIV-1 challenges in primary T-cells and macrophages and, importantly, demonstrates a survival and growth advantage of protected CD4 T-cells. The findings underscore the potential advantage of intrabody gene delivery to CD34 cells, which allows differentiation of protected T-cells, macrophages, and dendritic cells.

Making cell-permeable antibodies (Transbody) through fusion of protein transduction domains (PTD) with single chain variable fragment (scFv) antibodies: Potential advantages over antibodies expressed within the intracellular environment (Intrabody)

Over the past decade, there has been growing interest in the use of antibodies against intracellular targets. This is currently achieved through recombinant expression of the single chain variable fragment (scFv) antibody format within the cell, which is commonly referred to as an intrabody. This possesses a number of inherent advantages over RNA interference (iRNA). Firstly, the high specificity and affinity of intrabodies to target antigens is well-established, whereas iRNA has been frequently shown to exert multiple non-specific effects. Secondly, intrabodies being proteins possess a much longer active half-life compared to iRNA. Thirdly, when the active half-life of the intracellular target molecule is long, gene silencing through iRNA would be slow to yield any effect, whereas the effects of intrabody expression would be almost instantaneous. Lastly, it is possible to design intrabodies to block certain binding interactions of a particular target molecule, while sparing others. There is, however, various technical challenges faced with intrabody expression through the application of recombinant DNA technology. In particular, protein conformational folding and structural stability of the newly-synthesized intrabody within the cell is affected by reducing conditions of the intracellular environment. Also, there are overwhelming safety concerns surrounding the application of transfected recombinant DNA in human clinical therapy, which is required to achieve intrabody expression within the cell. Of particular concern are the various viral-based vectors that are commonly-used in genetic manipulation. A novel approach around these problems would be to look at the possibility of fusing protein transduction domains (PTD) to scFv antibodies, to create a 'cell-permeable' antibody or 'Transbody'. PTD are short peptide sequences that enable proteins to translocate across the cell membrane and be internalized within the cytosol, through atypical secretory and internalization pathways. There are a number of distinct advantages that a 'Transbody' would possess over conventional intrabodies expressed within the cell. For a start, 'correct' conformational folding and disulfide bond formation can take place prior to introduction into the target cell. More importantly, the use of cell-permeable antibodies or 'Transbodies' would avoid the overwhelming safety and ethical concerns surrounding the direct application of recombinant DNA technology in human clinical therapy, which is required for intrabody expression within the cell. 'Transbodies' introduced into the cell would possess only a limited active half-life, without resulting in any permanent genetic alteration. This would allay any safety concerns with regards to their application in human clinical therapy.