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Intrabodies as therapeutic agents
Methods 34 (2004) 163–170 www.elsevier.com/locate/ymeth
Intrabodies as therapeutic agents Roland E. Kontermann¤ Vectron therapeutics AG, Rudolf-Breitscheid-Str. 24, 35037 Marburg, Germany Accepted 8 April 2004 Available online 6 July 2004
Abstract In the past decade, a large number of intracellular antibodies (intrabodies) have been developed for potential use as therapeutic agents. As antibodies can be generated against virtually any target antigen, the applications of intrabodies span a wide range including tumour therapy, infectious diseases, transplantation, and other diseases associated with protein overexpression or mutagenesis. This article summarises the development of intrabodies and their applications as therapeutic agents. 2004 Elsevier Inc. All rights reserved.
1. Introduction
2. Directing intrabodies to subcellular compartments
Intrabodies (intracellular antibodies) are deWned as antibody molecules which are expressed intracellularly and directed to deWned subcellular compartments. Although mammalian cells are the most commonly used target cells, expression of intrabodies is not restricted to these cells and a variety of other cell types, including plant cells, fungal cells, and even bacteria, have been used. The therapeutic concept of using intrabodies is based on the induction of a phenotypic knockout of a relevant target molecule either by directly inhibiting the function of the antigen or by diverting it from its normal intracellular location. In some cases, intrabodies have also been used to restore the function of a target antigen and thus rescuing a phenotype. Thus, intrabody therapy combines the speciWcity of antibodies with a gene-therapeutic strategy to selectively aVect an intracellular target protein. In contrast to the direct administration of a therapeutic drug, this approach engages the cellular machinery to produce the therapeutic agent. As the intrabodies are produced only inside the cells, this strategy has advantages regarding safety and eYcacy.
To exert their function, intrabodies have to be directed to the subcellular compartment where the antigen is located. This can be achieved by the incorporation of signal sequences into the antibody molecules, routinely either fused to the N- or C-terminus. A variety of signal sequences are known which are able to direct the antibody molecules to subcellular compartments such as the nucleus, endoplasmic reticulum (ER), trans-Golgi network (TGN), mitochondria, and cell membrane (see Fig. 1). Alternatively, the expression of antibody molecules without any signal sequence retains the intrabodies in the cytoplasm ([1]; see Cardinale et al., this issue).
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3. Intracellular antibody formats The activity of intrabodies is due to binding to an intracellular target antigen. Thus, the antigen-binding site of antibodies is suYcient for this activity. Singlechain Fv fragments (scFvs) represent the recombinant minimal antigen-binding fragments of antibodies [2]. ScFvs are therefore the most commonly used format for intracellular expression. Their small size facilitates expression and assembly of functional molecules. However, intrabodies are not restricted to scFv fragments and various other formats including Fab fragments, scFv-C fusion proteins, single-chain diabodies, VH–CH1 fragments,
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Fig. 1. Subcellular location of intrabodies. Intrabodies have been expressed intracellularly and directed to the cytoplasm (1), mitochondria (2), the nucleus (3), the endoplasmic reticulum (4), the trans-Golgi network (TGN) (5), the plasma membrane (6), or secreted into the extracellular space (7).
and even whole IgG molecules have been applied (see Tables 1–3). An increased stability compared to scFvs was described for some of these formats. Although there are no comparative data available, the employment of dior multivalent intrabodies (e.g., bivalent diabodies or tandem scFv) should result in improved activity due to increased functional aYnity. In addition, the application of bispeciWc intrabodies should allow for an intracellular retargeting of molecules or the simultaneous phenotypic knockout of two target molecules [3]. One problem associated with expression of active intrabodies arises from the redox state of the subcellular com-
partments. As immunoglobulin domains including the variable antigen-binding domains are stabilised by intrachain disulphide bonds, production of functional antibodies requires a non-reducing milieu. This milieu is found in various compartments, such as the secretory compartments or mitochondria, and expression of intrabodies in these compartments results routinely in functional intrabodies [4]. In contrast, other compartments such as the cytoplasm and nucleus have a reducing milieu which can lead to incorrect folding and a reduced stability of intrabodies. To improve the stability of intrabodies, scFv scaVolds which fold into functional molecules in the cytoplasm
Table 1 Intrabodies for cancer therapy Target
Target function
Antibody format
Subcellular compartment
References
erbB2 EGFR BCR-ABL p21Ras Caspase 3 Caspase 7 Bcl-2 p53 Cyclin E ATF-1/CREB HPV16 E7 HP1 Type IV collagenases Cathepsin L
Signal transduction Signal transduction Signal transduction Signal transduction Apoptosis Apoptosis Anti-apoptotic Tumor suppression Cell cycle regulation Transcription activation Transcriptional regulation Heterochromatin assembly Matrix metalloproteinase Proteinase
scFv scFv scFv scFv, IgG scFv scFv scFv scFv scFv-Fc scFv scFv scFv scFv scFv
ER, cytoplasm Cytoplasm, ER ER Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm, nucleus Cytoplasm, nucleus Cytoplasm
[28–30,32–34,48–53] [31,48,54,55] [35] [4,19,20,36,56–67] [37,68] [38] [39] [40,65,69] [44] [41,42,70] [43] [45] [47] [46]
Cytoplasm ER ER
R.E. Kontermann / Methods 34 (2004) 163–170
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Table 2 Intrabodies for the treatment of HIV-1 infections Target
Target function
Antibody format
Subcellular compartment
References
HIV-1 gp120 HIV-1 gp41 CCR5
Viral coat protein Viral coat protein Chemokine receptor, HIV-1 infection HIV-1 co-receptor Reverse transcriptase Virus replication Viral RNA stability Integrase Transcription Matrix protein
scFv, Fab scFv scFv
ER ER, trans-Golgi ER
[72,73,79,80] [74] [81]
scFv scFv, VH–CH1 scFv-C scFv scFv scFv–Vpr scFv Fab, scFv, scFv-C
ER Cytoplasm Cytoplasm, nucleus Cytoplasm Cytoplasm, nucleus, virion Cytoplasm Cytoplasm, nucleus
[82] [83–85] [86–90] [75,91–95] [76,77,96] [78] [97–99]
CXCR4 HIV-1 RT HIV-1 Tat HIV-1 Rev HIV-1 IN Cyclin T1 HIV-1 p17
Table 3 Intrabodies for the treatment of other diseases Target
Target function
Antibody format
Subcellular compartment
References
Flavivirus envelope protein HCV NS3 Huntingtin IL-2R MHC-I 1,3-Galactosyl-transferase Root-knot nematode secretions Colicin A
Virus infectivity Non-structural 3 protein Mutated in Huntington’s disease Cytokine receptor Antigen presentation Glycosylation Nematode infection Bacteriotoxin
scFv scFv scFv scFv scFv scFv scFv scFv
Cytoplasm Cytoplasm Cytoplasm, nucleus ER ER ER ER Bacterial periplasm
[104] [105] [102,103] [106–108] [100] [101] [109] [110]
have been generated by genetic engineering and molecular evolution or by the employment of molecular chaperones [5–14]. Another approach is the direct in vivo selection or screening of stable antibody fragments from intrabody libraries. Such intrabodies are either identiWed by their ability to bind an antigen (intracellular antibody capture technology) ([15–17]; see also Cattaneo et al., this issue) or by an antigen-independent selection using a selectable marker fused to the scFv fragments ([18]; see also Maur et al., this issue). However, a reduced stability of cytoplasmic intrabodies does not necessarily mean that they are non-functional. A recent study of several anti-ras scFv intrabodies showed that expression of instable intrabodies in the cytoplasm leads to antigen-speciWc intracellular coaggregation of scFvs with its antigen. This indicates that the formation of such aggregates might be a general mode of action of cytoplasmic intrabodies for the generation of phenotypic knockouts [19,20].
ity is mediated by the intrabody, e.g., by targeting viral encoded or overexpressed/abnormal proteins, target-cell speciWcity of gene transfer is not essential, although it might inXuence therapeutic eYcacy. Gene transfer can be achieved by a variety of methods. Viral (e.g., retroviruses, adenoviruses, and adeno-associated viruses) and non-viral (e.g., liposomes, molecular conjugates) transfer systems are the most widely studied vehicles for gene transfer. Both systems have advantages and disadvantages associated with eYcacy, target cell speciWcity, non-speciWc uptake, immunogenicity, toxicity, and safety issues [23–25]. Regarding intrabodies, retroviral and adenoviral gene transfer systems have been developed to deliver intrabody genes into target cells ex vivo or in vivo [26,27]. These studies include one phase I trial for the treatment of ovarian cancer patients with an anti-erbB2 intrabody [28]. As described below this study demonstrated the need for highly eYcient gene transfer and/or the combination with strategies leading to bystander eVects in order to achieve a therapeutic beneWt.
4. Transfer of intrabody-encoding genes A key issue in the therapeutic use of intrabodies is the eYcient transfer of the intrabody-encoding gene into target cells. Target cell speciWcity of gene transfer is necessary if the intrabody targets a general pathway found in normal and diseased cells. Target cell speciWcity can be achieved either through transductional or transcriptional targeting [21,22]. On the other hand, if the speciWc-
5. Tumour therapy Antibodies are widely accepted therapeutic agents for the treatment of cancer, which is reXected by the growing number of approved antibodies and antibodies tested in preclinical and clinical studies. These antibodies are selected to act on extracellular targets after systemic
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or local administration. Functionally, these antibodies possess either neutralising activities or induce a humoral or cellular immune response leading to the destruction of tumour cells or the associated tumour vasculature. In addition, eYcacy can be increased by the conjugation of potent cytotoxic components such as drugs, toxins, or radionuclides. Antibodies can also be engaged as intrabodies aVecting intracellular targets and pathways associated with tumour cell proliferation, diVerentiation, and invasion. 5.1. Inhibition of receptor tyrosine kinases Receptor tyrosine kinases (RTKs) are regulators of cell growth and diVerentiation. Binding of a ligand to the extracellular domain results in receptor dimerisation and subsequent phosphorylation of intracellular substrates coupled to signal transduction pathways which regulate gene expression. Members of the epidermal growth factor RTK receptor family, e.g., EGF receptor and erbB2, are overexpressed by a variety of tumours including breast and ovary carcinoma. Intrabodies have been extensively studied as inhibitors of members of the EGF receptor family (see Table 1). Phenotypic knockout of EGFR or erbB2 was achieved by retention of the receptor molecules in the ER employing intrabodies equipped with an ER retention signal. This caused inhibition of tumour cell proliferation in vitro and in vivo and the eradication of the tumour cells by the induction of apoptosis [29–33]. Cytotoxic eVects could be correlated with the expression level of intrabodies and their target protein [34]. Antineoplastic eVects were also observed in animal models after adenovirus-mediated transfer of the intrabody-encoding gene into ovarian cancers [27]. This led to a phase I trial utilising an anti-erbB2 scFv-encoding adenovirus to treat ovarian cancer patients [28]. Although none of the patients treated showed dramatic clinical beneWts, due to gene transfer only into a limited number of tumour cells, this trial demonstrated the feasibility of this approach but highlighted also the need for eYcient gene transfer systems to transduce as many tumour cells as possible. The approach to inhibit the function of receptor tyrosine kinases by intrabodies is also applicable to other receptors than members of the EGF receptor family. Recently, novel intrabodies against the oncogenic protein BCR-ABL have been selected by an intracellular capture technology [35]. 5.2. Intrabodies inhibiting intracellular signal transduction pathways The Ras protein is a guanine nucleotide-binding protein which catalyses the hydrolysis of GTP to GDP. Its biochemical function depends on its GTP-binding and catalytic activity and on the correct cellular location at
the inner surface of the plasma membrane. Ras plays a central role in the signal transduction cascade of receptor tyrosine kinases acting on various targets (PI3kinase, Raf, and Ral). Overexpression or mutations in the ras gene are associated with a transformed phenotype of a variety of tumours leading to disregulation of cell growth and diVerentiation. Raf-1, a serine–threonine kinase, is one of the important down-stream eVectors of Ras function. Complexation of Ras with Raf-1, normally found in the cytoplasm, leads to translocation of Raf-1 to the plasma membrane and subsequent activation of its kinase function. The correct localisation of Ras to the inner surface of the plasma membrane is crucial for its function in stimulation of cellular proliferation. Studies with anti-ras intrabodies have shown that diverting Ras from the inner compartment of the plasma membrane, e.g., by forming cytoplasmic aggresomes, results in inhibition of its function (see Table 1). Furthermore, neutralisation of ras with a scFv intrabody derived from antibody Y13-259 promoted apoptosis in human cancer cells and led to tumour regression of a colon carcinoma tumour model in nude mice [36]. 5.3. The apoptotic pathway as target for intrabodies Programmed cell death (apoptosis) is an important process controlling the equilibrium of loss and growth of cells in an organism and the elimination of damaged or transformed cells. Programmed cell death is regulated by the interplay of pro-apoptotic and anti-apoptotic proteins. Apoptosis is carried out by a family of cysteine proteinases (caspases). A central role in this process plays the executioner caspase 3. Caspase 3 is activated by upstream caspases leading to the formation of an active tetrameric enzyme. It has been shown that forced dimerisation can cause self-activation of capase 3 and the induction of cell death. A scFv-fusion protein consisting of an anti--galactosidase scFv fused to caspase 3 was able to bring two caspase 3 domains in close proximity when co-expressed intracellularly with -galactosidase [37]. This led to autoactivation and triggering of apoptosis. Importantly, activation depended on dimerisation and the scFv–caspase fusion protein was non-toxic in normal cells. For tumour therapy, proteins such as p53 mutants or oncogenic chimeric fusion proteins resulting from chromosomal translocations may be candidate targets for the scFv–caspase 3 approach. Although applicability has not yet been demonstrated in such a setting, the use of intracellular scFv-fusion proteins which can activate an eVector function upon scFv-mediated dimerisation opens up a new way to regulate pathways involved in cell viability or tumourigenicity. Other studies of targeting the apoptotic pathway with intrabodies described the use of anti-caspase 7 as antigen, another proapoptotic proteinase [38]. Furthermore, intrabodies inhibiting the anti-apoptotic protein Bcl-2 enhanced
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drug-mediated cytotoxicity of Bcl-2 overexpressing tumour cells [39]. 5.4. Nuclear targets of intrabodies Intrabodies targeting nuclear targets have been described to induce growth arrest and cell death. These targets include cell cycle regulators, p53, transcription factors, viral and cellular oncogenic proteins, or structural components of the chromatin (see Table 1). Intrabodies which are able to restore the DNA-binding and therefore transcriptional activity of certain p53 mutants have recently been described [40]. These intrabodies are one of the few examples where an antibody is able to revert an inactive mutant back into a functional molecule. Such intrabodies may represent a novel class of molecules for p53-based cancer therapy. Upregulation of transcription factors is often associated with malignancies. The heterodimeric transcription factor ATF-1/CREB, involved in cAMP and Ca2+ induced transcriptional activity, is upregulated in metastatic melanoma cells [41,42]. An intrabody inhibiting binding of ATF-1 to DNA was able to reduce CRE-dependent promoter activity and to suppress tumourigenicity and metastatic potential in a melanoma cancer animal model. The viral oncoprotein E7 of the human papillomavirus type 16 is found in over 90% of cervical cancers and plays a major role in tumourigenicity through interaction with several proteins involved in transcription and cell cycle regulation, e.g., the retinoblastoma protein pRb. An intrabody directed against E7 was able to down-regulate E7 and to inhibit cell proliferation in vitro [43]. Cell cycle regulation has been targeted with an intrabody directed against cyclin E [44]. Cyclin E plays a central role in regulating cell cycle progression from G1 to S phase in normal cells. Dysregulated cyclin E is found in nearly all breast cancers and leads to an accelerated cell cycle and chromosome instability. The anti-cyclin E intrabody was expressed as scFv–Fc fusion protein resulting in a dramatic increase in half-life (see also Strube and Chen; this issue). Targeting of the intrabody to the nucleus caused growth inhibition of a breast cancer cell line. This indicates that inhibiting cell cycle regulation might be a potential target for cancer therapy. Heterochromatin protein 1 (HP1) is involved in silencing gene expression, nuclear envelope re-assembly, recruiting of proteins to the mitotic spindle, and spermatogenesis. An intrabody directed against the chromodomain (CD) of HP1 triggered a p53-independent apoptotic pathway leading to a cell-lethal phenotype in mouse or human Wbroblasts [45]. 5.5. Extracellular proteinases as targets Various proteinases expressed by transformed cells are involved in tumour progression or play a central role
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in tumour invasion and metastasis formation. Cathepsin L, a cysteine proteinase, is found intracellularly in lysosomes where it degrades a wide range of cytoplasmic and nuclear proteins. Cathepsin L is also overexpressed and secreted by tumour cells, e.g., melanoma cells contributing to tumour progression. An intrabody directed against the procathepsin L form was shown to cause inhibition of secretion and intracellular accumulation of the protein [46]. Although inhibition of tumour growth has not been demonstrated, anti-cathepsin L intrabodies may be useful tools to inhibit the tumourigenic and metastatic phenotype of human melanoma cells. Another type of proteinases, the metalloproteinases MMP-2 and MMP-9 which both degrade collagenase IV in the basement membrane, play an important role in tumour invasion and the formation of metastasis. Expression of anti-type IV collagenase intrabody in the ER of a highly metastatic lung carcinoma cell line resulted in down-regulation of collagenase expression and the inhibition of invasion in an in vitro colony formation assay [47]. Such intrabodies directed against tumour-associated proteinases may represent a novel approach for the inhibition of tumour invasion in vivo.
6. Treatment of infectious diseases 6.1. HIV-1 Over 40 million people were infected with the human immunodeWciency virus 1 (HIV-1) at the end of 2002 with a lethality of approximately 7%. HIV-1 mainly infects T cells and macrophages leading to immunodeWciency syndromes. The CD4 receptor and various co-receptors on the target cell surface mediate virus absorption and infection. The virus encodes a total of 15 distinct proteins including structural proteins or proteins involved in virus replication, gene regulation, processing of viral proteins and the genome, and virus assembly [71]. All these proteins are potential targets for intrabodies. Intrabodies directed against several of these proteins including the viral coat proteins gp120, and gp41, the matrix protein p17, proteins involved in transcription and replication (reverse transcriptase, Tat, Rev) or integration of the viral DNA into the host genome (integrase) have been described (see Table 2). These intrabodies eYciently inhibited HIV-1 production in host cells. Intrabodies directed against gp120 were able to inhibit virus production when expressed in the ER. This approach was extended to a combined intra- and extracellular immunisation using the same antibody to block binding of the virus to CD4 [72]. Lymphocytes transduced with the intrabody expression cassette using an adeno-associated virus shuttle vector did not show any cytotoxicity and were resistant to infection by several primary HIV-1 patient isolates [73]. In another study,
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non-neutralising anti-gp41 intrabodies directed to the ER or trans-Golgi network bound to gp160 and inhibited HIV-1 replication. Intrabodies expressed in the ER were able to block processing of gp160 into gp120 and gp41, in contrast to intrabodies localised in the trans-Golgi network, indicating that the intrabodies use diVerent mechanisms to inhibit HIV-1 replication [74]. Importantly, this study showed that non-neutralising antibodies are useful therapeutic agents when expressed intracellularly. Most neutralising antibodies are directed against epitopes which show a high rate of mutation, while epitopes recognised by non-neutralising antibodies are often more conserved among virus isolates. Thus, the use of such non-neutralising intrabodies may represent an advantage for the development of intrabodies to Wght HIV-1. The replication of viral DNA was targeted with cytoplasmic or nuclear intrabodies inhibiting the reverse transcriptase or the transactivators Tat or Rev (see Table 2). The use of Rev as target has several advantages as it is essential for posttranscriptional processing of the initial viral transcript, is speciWc for a highly conserved Rev-responsive element (RRE), and exhibits favourable threshold kinetics [75]. The inhibitory activity of antiRev intrabodies was further improved by combining them with an RRE decoy RNA targeting both the Rev protein and Rev-binding site which resulted in eYcient inhibition of infection by various virus isolates. Another target is the HIV-1 integrase (IN) packaged into the virus particle and involved in virus replication. Cytoplasmic or nuclear expression of anti-IN intrabodies recognising the catalytic or C-terminal domains of IN resulted in resistance to productive HIV-1 infection [76]. In another approach, an anti-integrase scFv was expressed in fusion with the viral accessory protein Vpr which is packaged into the virion in high quantities. The fusion protein was eYciently encapsidated into the virion resulting in a decreased infectivity. Thus, this intra-virion gene therapy approach, which targets virus replication as well as virion spread, leads to increased inhibitory eVects [77]. Targeting of host proteins involved in HIV-1 infection and/or replication represents another approach for HIV-1 therapy as phenotypic knockout of HIV-1 receptors or co-receptors should lead to inhibition of HIV-1 infection. Two coreceptors, CXCR4 and CCR5, have been demonstrated to be relevant for HIV-1 cell entry in vivo. While CCR5 is involved in the primary infection with R5 viruses, disease progression correlates with the evolvement of X4 strains which use CXCR4 as major co-receptor. Both receptors have been targeted with intrabodies expressed in the ER (see Table 2) causing functional deletion or down-regulation of the receptor and resistance to viral infection. Other targets are host proteins necessary for virus replication. One such protein is human cyclin T1 (hCyclinT1) which is a subunit of the positive transcription elongation factor (P-TEFb)
complex. This protein interacts with Tat and binds cooperatively to the transactivation response element (TAR) leading to transactivation of HIV-1 transcription. AntihCyclinT1 intrabodies directed against the cyclin box domain were able to block HIV-1 replication. Importantly, they did not cause cellular toxicity making these intrabodies useful agents for HIV-1 therapy [78]. 6.2. Other infectious diseases Intrabodies have also been described for the treatment of various other viral infections including the inhibition of tick-borne Xavivirus infection with a neutralising intrabody directed against the envelope protein and the inhibition of hepatitis C virus (HVC) replication with an intrabody recognising the non-structural 3 protein (NS3) (see Table 3). Although as of yet only few intrabody approaches have been described for the therapy of bacterial or parasite infections (see Table 3), intrabodies should have the potential to treat these diseases by targeting the entry or replication/assembly pathway or by directly neutralising toxicity of a target protein.
7. Intracellular immune suppression Allocraft rejection is caused by cellular as well as humoural components of the immune system and the inhibition of these events is essential for a successful transplantation. One of the central processes leading to rejection is the recognition of MHC class I antigens of the donor cells by host T cells. In a recent study, antiMHC-I heavy chain intrabodies directed to the ER were described to induce marked down-regulation of MHC-I surface expression in adenovirus transduced primary human keratinocytes, even in the presence of MHC-I upregulating cytokines [100]. MHC-I knockout might Wnd applications in impairing alloantigen expression and prolonging the survival of allografts. Another approach used an intrabody against the enzyme 1,3-galactosyltransferase to inhibit 1,3-galactosylation of cell surface antigens [101]. This carbohydrate structure is a major antigen recognised by xenoreactive natural antibodies in higher primates. The intrabody was able to reduce cytotoxicity mediated by anti-Gal- 1,3Gal antibodies in vitro by more than 90%. Although the enzymatic activity was not completely knocked out, such intrabodies may be useful to protect cells from elicited antibodies or innate eVectors.
8. Other applications Intrabodies may also Wnd applications for the treatment of diseases associated with the expression of altered proteins responsible for a pathogenic phenotype which
R.E. Kontermann / Methods 34 (2004) 163–170
are normally not found in healthy cells. One such example is the Huntington’s disease (HD), a neurodegenerative disorder caused by extended polyglutamine stretches of the huntingtin protein. Stretches of more than 40 glutamines are lethal in subpopulations of neurons in the striatum and cortex, which is associated with the formation of insoluble aggregates in aVected neurons. The in vitro analysis of various intrabodies recognising diVerent regions of the huntingtin protein showed that intrabodies directed against a polyproline stretch or the N-terminus are able to inhibit aggregation as well as cell death while anti-polyglutamine intrabodies stimulated aggregation and apoptosis [102,103]. Such inhibitory intrabodies may be of therapeutic value in the treatment of HD.
9. Concluding remarks Although far from being clinically approved therapeutic agents, intrabodies extend the application of antibodies by targeting antigens not accessible by circulating antibody molecules. The development of intracellularly expressed antibodies introduced a novel way of antigen neutralisation by diverting it from its normal subcellular location causing a phenotypic knockout. As summarised in this article, a large number of diVerent applications have already been established for intrabodies. Certainly, in the future intrabodies will be developed against additional targets and employed for the treatment of a variety of other diseases. However, there remain problems to be solved including improved expression and stability of intrabodies and the development of eYcient and safe gene transfer systems.
References [1] S. Biocca, A. Cardinale, A. Cataneo, in: R.E. Kontermann, S. Dübel (Eds.), Antibody Engineering, Springer Heidelberg Press, New York, 2001, pp. 755–774. [2] P.J. Hudson, Curr. Opin. Biotechnol. 9 (1998) 395–402. [3] R.E. Kontermann, R. Müller, J. Immunol. Methods 226 (1999) 179–188. [4] S. Biocca, F. Ruberti, M. Tafani, P. Pierandrei-Amaldi, A. Cattaneo, Bio/Technology 13 (1995) 1110–1115. [5] P. Jurado, d. Ritz, J. Beckwith, V. de Lorenzo, L.A. Fernandez, J. Mol. Biol. 320 (2002) 1–10. [6] J.C. Laden, P. Philibert, F. Torreilles, M. Pugniere, P. Martineau, Res. Microbiol. 153 (2002) 469–474. [7] P. Martineau, J.M. Betton, J. Mol. Biol. 292 (1999) 921–929. [8] P. Martineau, P. Jones, G. Winter, J. Mol. Biol. 280 (1998) 117–127. [9] E.C. Ohage, B. Steipe, J. Mol. Biol. 291 (1999) 1119–1128. [10] P. Writz, B. Steipe, Protein Sci. 8 (1999) 2245–2250. [11] E.C. Ohage, P. Writz, J. Barnikow, B. Steipe, J. Mol. Biol. 291 (1999) 1129–1134. [12] A. Wörn, A. Auf der Maur, D. Escher, A. Honegger, A. Barberis, A. Plückthun, J. Biol. Chem. 275 (2000) 2795–2803. [13] K. Proba, A. Wörn, A. Honegger, A. Plückthun, J. Mol. Biol. 275 (1998) 245–253. [14] H. Bach, Y. Mazor, S. Shaky, A. Shoham-Lev, Y. Berdichevsky, D.L. Gutnick, I. Benhar, J. Mol. Biol. 312 (2001) 79–93.
169
[15] M. Visintin, E. Tse, H. Axelson, T.H. Rabbitts, A. Cattaneo, Proc. Natl. Acad. Sci. USA 96 (1999) 11723–11728. [16] T. Tanaka, G.T. Chung, A. Forster, M.N. Lobato, T.H. Rabbitts, Nucleic Acids Res. 31 (2003) e23. [17] A. auf der Maur, C. Zahnd, F. Fischer, S. Spinelli, A. Honegger, C. Cambillau, D. Escher, A. Plückthun, A. Barberis, J. Biol. Chem. 277 (2002) 45075–45085. [18] A. auf der Maur, D. Escher, A. Barberis, FEBS Lett. 508 (2001) 407–412. [19] M. Lener, I.R. Horn, A. Cardinale, S. Mesina, U.B. Nielsen, S.M. Rybak, H.R. Hoogenboom, A. Cattaneo, S. Biocca, Eur. J. Biochem. 267 (2000) 1196–1205. [20] A. Cardinale, I. Filesi, S. Biocca, Eur. J. Biochem. 268 (2001) 268– 277. [21] K.-W. Peng, S.J. Russell, Curr. Opin. Biotechnol. 10 (1999) 454– 457. [22] D.V. SchaVer, D.A. LauVenburger, Curr. Opin. Mol. Ther. 2 (2000) 155–161. [23] I. Verma, N. Somia, Nature 389 (1997) 239–242. [24] F. Meyer, M. Finer, Cell. Mol. Biol. 47 (2001) 1277–1294. [25] A. Mountain, Trends Biotechnol. 18 (2000) 119–128. [26] D. Zhan, A. Murakami, R.P. Johnson, J. Sui, J. Cheng, J. Bai, W.A. Marasco, J. Acquir. Immune DeWc. Syndr. 32 (2003) 245– 254. [27] J. Deshane, G.P. Siegal, M. Wang, M. Wright, R.P. Bucy, R.D. Alvarez, D.T. Curiel, Gynecol. Oncol. 64 (1997) 378–385. [28] R.D. Alvarez, M.N. Barnes, J. Gomez-Navarro, M. Wang, T.V. Strong, W. Arafat, R.B. Arani, M.R. Johnson, B.L. Roberts, G.P. Siegal, D.T. Curiel, Clin. Cancer Res. 6 (2000) 3081–3087. [29] R.R. Beerli, W. Wels, N.E. Hynes, J. Biol. Chem. 269 (1994) 23931–23936. [30] J. Deshane, F. Loechel, R.M. Conry, G.P. Siegal, C.R. King, D.T. Curiel, Gene Ther. 1 (1994) 332–337. [31] C.B. Jannot, R.R. Beerli, S. Mason, W.J. Gullick, N.E. Hynes, Oncogene 13 (1996) 275–282. [32] J. Deshane, G. Cabrera, J.E. Grim, G.P. Siegal, J. Pike, R.D. Alvarez, D.T. Curiel, Gynecol. Oncol. 59 (1995) 8–14. [33] J. Deshane, J . Grim, S. Loechel, G.P. Siegal, R.D. Alvarez, D.T. Curiel, Cancer Gene Ther. 3 (1996) 89–98. [34] J. Grim, J. Deshane, G.P. Siegal, R.D. Alvarez, P. DiFiore, D.T. Curiel, J. Mol. Med. 76 (1998) 451–458. [35] E. Tse, M.N. Lobato, A. Forster, T. Tanaka, G.T. Chung, T.H. Rabbitts, J. Mol. Biol. 317 (2002) 85–94. [36] O. Cochet, M. Kenigsberg, I. Delumeau, A. Vvirone-Oddos, M.C. Multon, W.H. Fridman, F. SchweighoVer, J.L. Teillaud, B. Tocques, Cancer Res. 58 (1998) 1170–1176. [37] E. Tse, T.H. Rabbitts, Proc. Natl. Acad. Sci. USA 97 (2000) 12266–12271. [38] Q. Zhu, C. Zeng, A. Huhalov, J. Yao, T.G. Turi, D. Danley, T. Hynes, Y. Cong, D. DiMattia, S. Kennedy, G. Daumy, E. SchaeVer, W.A. Marasco, J.S. Huston, J. Immunol. Methods 231 (1999) 207–222. [39] A. Piche, J. Grim, C. Rancourt, J. Gomez-Navarro, J.C. Reed, D.T. Curiel, Cancer Res. 58 (1998) 2134–2140. [40] C. de Fromentel, N. Gruel, C. Venot, L. Debussche, E. Conseiller, C. Dureuil, J.L. Teillaud, B. Tocque, L. Bracco, Oncogene 18 (1999) 551–557. [41] D. Jean, M. Bar-Eli, Crit. Rev. Immunol. 21 (2001) 275–286. [42] D. Jean, C. Tellez, S. Huang, D.W. Davis, C.J. Bruns, D.J. McConkey, S.H. Hinrichs, M. Bar-Eli, Oncogene 19 (2000) 2721– 2730. [43] F. Wang-Johanning, G.Y. Gillespie, J. Grim, C. Rancourt, R.D. Alvarez, G.P. Siegal, D.T. Curiel, Cancer Res. 58 (1998) 1893–1900. [44] R.W. Strube, S.-Y. Chen, J. Immunol. Methods 263 (2002) 149– 167. [45] I. Filesi, A. Cardinale, S. van der Sar, I.G. Cowell, P.B. Singh, S. Biocca, J. Cell Sci. 115 (2002) 1803–1813.
170
R.E. Kontermann / Methods 34 (2004) 163–170
[46] N. Guillaume-Rousselet, D. Jean, R. Frade, Biochem. J. 367 (2002) 219–227. [47] W. Wang, J. Zhou, L. Xu, Y. Zhen, J. Environ. Pathol. Toxicol. Oncol. 19 (2000) 61–68. [48] R.R. Beerli, W. Wels, N.E. Hynes, Breast Cancer Res. Treat. 38 (1996) 11–17. [49] J. Deshane, G.P. Siegal, R.D. Alvarez, M.H. Wang, M. Feng, M. Feng, G. Cabrera, T. Liu, M. Kay, D.T. Curiel, J. Clin. Invest. 96 (1995) 2980–2989. [50] D. Graus-Porta, R.R. Beerli, N.E. Hynes, Mol. Cell. Biol. 15 (1995) 1182–1191. [51] M. Wright, J. Grim, J. Deshane, M. Kim, T.V. Strong, G.P. Siegal, D.T. Curiel, Gene Ther. 4 (1997) 317–322. [52] J. Grim, J. Deshane, M. Feng, A. Lieber, M. Kay, D.T. Curiel, Am. J. Respir. Cell. Mol. Biol. 15 (1996) 348–354. [53] W. Arafat, J. Gomez-Navarro, J. Xiang, G.P. Siegal, R.D. Alvarez, D.T. Curiel, Cancer Gene Ther. 7 (2000) 1250–1256. [54] R.R. Beerli, W. Wels, N.E. Hynes, Biochem. Biophys. Res. Commun. 204 (1994) 666–672. [55] S. Hyland, R.R. Beerli, C.F. Barbas III, N.E. Hynes, W. Wels, Oncogene 22 (2003) 1557–1567. [56] S. Biocca, M.S. Neuberger, A. Cattaneo, EMBO J. 9 (1990) 101–108. [57] S. Biocca, A. Di Luzio, T. Werge, A. Cattaneo, Cytotechnology 5 (Suppl. 1) (1991) 49–50. [58] S. Biocca, P. Perandrei-Amaldi, A. Cattaneo, Biochem. Biophys. Res. Commun. 197 (1993) 422–427. [59] S. Biocca, P. Perandrei-Amaldi, N. Campioni, A. Cattaneo, Bio/ Technology 12 (1993) 396–399. [60] A. Cardinale, M. Lener, S. Messina, A. Cattaneo, S. Biocca, FEBS Lett. 439 (1998) 197–202. [61] T.M. Werge, S. Biocca, A. Cattaneo, FEBS Lett. 274 (1990) 193–198. [62] T.M. Werge, C.T. Baldari, J.L. Telford, FEBS Lett. 351 (1994) 393–396. [63] X. Montano, A. Jimenez, Cell Growth DiV. 6 (1995) 597–605. [64] O. Cochet, M. Kenigsberg, I. Delumeau, M. Duchesne, F. SchweighoVer, B. Tocque, J.L. Teillaud, Mol. Immunol. 35 (1998) 1097–1110. [65] O. Cochet, N. Gruel, W.H. Frisman, J.L. Teillaud, Cancer Detect. Prev. 23 (1999) 506–510. [66] T. Tanaka, T.H. Rabbitts, EMBO J. 22 (2003) 1025–1035. [67] E. Tse, G. Chung, T.H. Rabbitts, Methods Mol. Biol. 185 (2002) 433–446. [68] A. Rajpal, T.G. Turi, J. Biol. Chem. 276 (2001) 33139–33146. [69] P.A. Cohen, J.C. Mani, D.P. Lane, Oncogene 17 (1998) 2445–2456. [70] J.M. Bosilevac, C.A. Gilchrist, P.E. Jankowski, S. Paul, A.R. Rees, S.H. Hinrichs, J. Biol. Chem. 273 (1998) 16874–16879. [71] A.D. Frankel, J.A. Young, Annu. Rev. Biochem. 67 (1998) 1–25. [72] S.-Y. Chen, Y. Khouri, J. Bagley, W.A. Marasco, Proc. Natl. Acad. Sci. USA 91 (1994) 5932–5936. [73] J.D. Chen, Q. Yang, A.G. Yang, W.A. Marasco, S.-Y. Chen, Hum. Gene Ther. 7 (1996) 1515–1525. [74] P. Zhou, S. Goldstein, K. Devadas, D. Tewari, A.L. Notkins, J. Immunol. 160 (1998) 14889–14896. [75] R.T. Inouye, B. Du, D. Boldt-Houle, A. Ferrante, I.-W. Park, S.M. Hammer, L. Duan, J.E. Groopman, R.J. Pomerantz, E.F. Terwilliger, J. Virol. 71 (1997) 4071–4078. [76] P. Levy-Mintz, L. Duan, H. Zhang, B. Hu, G. Dornadula, M. Zhu, J. Kulkosky, D. Bizub-Bender, A.M. Skalka, R.J. Pomerantz, J. Virol. 70 (1996) 8821–8832. [77] M. BouHamdan, J. Kulkosky, L.X. Duan, R.J. Pomerantz, J. Hum. Virol. 3 (2000) 6–15. [78] J. Bai, J. Sui, R.Y. Zhu, A.S. Tallarico, F. Gennari, D. Zhang, W.A. Marasco, J. Biol. Chem. 278 (2003) 1433–1442. [79] W.A. Marasco, W.A. Haseltine, S.-Y. Chen, Proc. Natl. Acad. Sci. USA 90 (1993) 7889–7893. [80] S.-Y. Chen, J. Bagley, W.A. Marasco, Hum. Gene Ther. 5 (1994) 595–601.
[81] P. Steinberger, J . Andris-Widhopf, B . Buhler, B.E. Torbett, C.F. Barbas III, Proc. Natl. Acad. Sci. USA 97 (2000) 805–810. [82] M. BouHamdan, D.S. Strayer, D. Wei, M. Mukhtar, L.X. Duan, J. Hoxie, R.J. Pomerantz, Gene Ther. 8 (2001) 408–418. [83] F. Shaheen, L. Duan, M. Zhu, O. Bagasra, R.J. Pomerantz, J. Virol. 70 (1996) 3382–4000. [84] N. Gargano, S. Biocca, A. Bradbury, A. Cattaneo, J. Virol. 70 (1996) 7706–7712. [85] N. Gargano, A. Cattaneo, J. Gen. Virol. 78 (1997) 2591–2599. [86] A.M. Mhashilkar, J. Bagley, S.-Y. Chen, A.M. Szilvay, D.G. Helland, W.A. Marasco, EMBO J. 14 (1995) 1542–1551. [87] A.M. Mhashilkar, D.K. Biswas, J . LaVecchio, A.B. Pardee, W.A. Marasco, J. Virol. 71 (1997) 6486–6494. [88] A.M. Mhashilkar, J. LaVecchio, B. Eberhardt, J. Porter-Brooks, S. Boisot, J.H . Dove, C. Pumphrey, X. Li, R.N. Weissmahr, D.B. Ring, U. Ramstedt, W.A. Marasco, Hum. Gene Ther. 10 (1999) 1453–1467. [89] W.A. Marasco, J. LaVecchio, A. Winkler, J. Immunol. Methods 231 (1999) 223–238. [90] M.C. Poznansky, J. La Vecchio, S. Silva-Arietta, J. PorterBrooks, K. Brody, I.T. Olszak, G.B. Adams, U. Ramstedt, W.A. Marasco, D.T. Scadden, Hum. Gene Ther. 10 (1999) 2505–2514. [91] L. Duan, O. Bagasra, M.A. Laughlin, J.W. Oakes, R.J. Pomerantz, Proc. Natl. Acad. Sci. USA 91 (1994) 5057–5079. [92] L.X. Duan, H. Zhang, J.W. Oakes, O. Bagasra, R.J. Pomerantz, Hum. Gene Ther. 5 (1994) 1315–1324. [93] L.X. Duan, M. Zhu, O. Bagasra, R.J. Pomerantz, Hum. Gene Ther. 6 (1995) 1561–1571. [94] L. Duan, M. Zhu, I. Ozaki, H. Zhang, D.L. Wei, R.J. Pomerantz, Gene Ther. 4 (1997) 533–543. [95] Y. Wu, L. Duan, M. Zhu, B. Hu, S. Kubota, O. Bagasra, R.J. Pomerantz, J. Virol. 70 (1996) 3290–3297. [96] M. BouHamdan, L.X. Duan, R.J. Pomerantz, D.S. Strayer, Gene Ther 6 (1999) 660–666. [97] R. Levin, A.M. Mhashilkar, T. Dorfman, A. Bukovsky, C. Zani, J. Bagley, J. Hinkula, M. Niedrig, J. Albert, B. Wahren, H.G. Gottlinger, W.A. Marasco, Mol. Med. 3 (1997) 96–110. [98] D. Tewari, S.L. Goldstein, A.L. Notkins, P. Zhou, J. Immunol. 161 (1998) 2642–2647. [99] D. Tewari, A.L. Notkins, P. Zhou, J. Gene Med. 5 (2003) 182– 189. [100] A.M. Mhashilkar, C. Doebis, M. Seifert, A. Busch, C. Zani, J. Soo Hoo, M. Nagy, T. Ritter, H.D. Volk, W.A. Marasco, Gene Ther. 9 (2002) 307–319. [101] B. Vanhove, B. Charreau, A. Cassard, C. Pourcel, J.P. Soulilou, Transplantation 66 (1998) 1477–1485. [102] A. Khoshnan, J. Ko, P.H. Patterson, Proc. Natl. Acad. Sci. USA 99 (2002) 1002–1007. [103] J.M. Lecerf, T.L. Shirley, Q. Zhu, A. Kazantsev, P. Amersdorfer, D.E. Housman, A. Messer, J.S. Huston, Proc. Natl. Acad. Sci. USA 98 (2001) 4764–4769. [104] W. Jiang, K. Venugopal, E.A. Gould, J. Virol. 69 (1995) 1044– 1049. [105] D.E. Sullivan, M.U. Mondelli, D.T. Curiel, V. Krasnykh, G. Mikheeva, P. Gaglio, C.B. Morris, S. Dash, M.A. Gerber, J. Hepatol. 37 (2002) 660–668. [106] J.H. Richardson, J.G. Sodroski, T.A. Waldmann, W.A. Marasco, Proc. Natl. Acad. Sci. USA 92 (1995) 3137–3141. [107] J.H. Richardson, T.A. Waldmann, J.G. Sodroski, W.A. Marasco, Virology 237 (1997) 209–216. [108] J.H. Richardson, W. Hofmann, J.G. Sodroski, W.A. Marasco, Gene Ther. 5 (1998) 635–644. [109] M.N. Rosso, A. Schouten, J. Roosien, T. Borst-Vrenssen, R.S. Hussey, F.J. Gommers, J. Bakker, A. Schots, P. Abad, Biochem. Biophys. Res. Commun. 220 (1996) 255–263. [110] P. Chames, J. Fieschi, D. Baty, D. Duche, J. Bacteriol. 180 (1998) 514–518.
Intrabodies as therapeutic agents Roland E. Kontermann¤ Vectron therapeutics AG, Rudolf-Breitscheid-Str. 24, 35037 Marburg, Germany Accepted 8 April 2004 Available online 6 July 2004
Abstract In the past decade, a large number of intracellular antibodies (intrabodies) have been developed for potential use as therapeutic agents. As antibodies can be generated against virtually any target antigen, the applications of intrabodies span a wide range including tumour therapy, infectious diseases, transplantation, and other diseases associated with protein overexpression or mutagenesis. This article summarises the development of intrabodies and their applications as therapeutic agents. 2004 Elsevier Inc. All rights reserved.
1. Introduction
2. Directing intrabodies to subcellular compartments
Intrabodies (intracellular antibodies) are deWned as antibody molecules which are expressed intracellularly and directed to deWned subcellular compartments. Although mammalian cells are the most commonly used target cells, expression of intrabodies is not restricted to these cells and a variety of other cell types, including plant cells, fungal cells, and even bacteria, have been used. The therapeutic concept of using intrabodies is based on the induction of a phenotypic knockout of a relevant target molecule either by directly inhibiting the function of the antigen or by diverting it from its normal intracellular location. In some cases, intrabodies have also been used to restore the function of a target antigen and thus rescuing a phenotype. Thus, intrabody therapy combines the speciWcity of antibodies with a gene-therapeutic strategy to selectively aVect an intracellular target protein. In contrast to the direct administration of a therapeutic drug, this approach engages the cellular machinery to produce the therapeutic agent. As the intrabodies are produced only inside the cells, this strategy has advantages regarding safety and eYcacy.
To exert their function, intrabodies have to be directed to the subcellular compartment where the antigen is located. This can be achieved by the incorporation of signal sequences into the antibody molecules, routinely either fused to the N- or C-terminus. A variety of signal sequences are known which are able to direct the antibody molecules to subcellular compartments such as the nucleus, endoplasmic reticulum (ER), trans-Golgi network (TGN), mitochondria, and cell membrane (see Fig. 1). Alternatively, the expression of antibody molecules without any signal sequence retains the intrabodies in the cytoplasm ([1]; see Cardinale et al., this issue).
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3. Intracellular antibody formats The activity of intrabodies is due to binding to an intracellular target antigen. Thus, the antigen-binding site of antibodies is suYcient for this activity. Singlechain Fv fragments (scFvs) represent the recombinant minimal antigen-binding fragments of antibodies [2]. ScFvs are therefore the most commonly used format for intracellular expression. Their small size facilitates expression and assembly of functional molecules. However, intrabodies are not restricted to scFv fragments and various other formats including Fab fragments, scFv-C fusion proteins, single-chain diabodies, VH–CH1 fragments,
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Fig. 1. Subcellular location of intrabodies. Intrabodies have been expressed intracellularly and directed to the cytoplasm (1), mitochondria (2), the nucleus (3), the endoplasmic reticulum (4), the trans-Golgi network (TGN) (5), the plasma membrane (6), or secreted into the extracellular space (7).
and even whole IgG molecules have been applied (see Tables 1–3). An increased stability compared to scFvs was described for some of these formats. Although there are no comparative data available, the employment of dior multivalent intrabodies (e.g., bivalent diabodies or tandem scFv) should result in improved activity due to increased functional aYnity. In addition, the application of bispeciWc intrabodies should allow for an intracellular retargeting of molecules or the simultaneous phenotypic knockout of two target molecules [3]. One problem associated with expression of active intrabodies arises from the redox state of the subcellular com-
partments. As immunoglobulin domains including the variable antigen-binding domains are stabilised by intrachain disulphide bonds, production of functional antibodies requires a non-reducing milieu. This milieu is found in various compartments, such as the secretory compartments or mitochondria, and expression of intrabodies in these compartments results routinely in functional intrabodies [4]. In contrast, other compartments such as the cytoplasm and nucleus have a reducing milieu which can lead to incorrect folding and a reduced stability of intrabodies. To improve the stability of intrabodies, scFv scaVolds which fold into functional molecules in the cytoplasm
Table 1 Intrabodies for cancer therapy Target
Target function
Antibody format
Subcellular compartment
References
erbB2 EGFR BCR-ABL p21Ras Caspase 3 Caspase 7 Bcl-2 p53 Cyclin E ATF-1/CREB HPV16 E7 HP1 Type IV collagenases Cathepsin L
Signal transduction Signal transduction Signal transduction Signal transduction Apoptosis Apoptosis Anti-apoptotic Tumor suppression Cell cycle regulation Transcription activation Transcriptional regulation Heterochromatin assembly Matrix metalloproteinase Proteinase
scFv scFv scFv scFv, IgG scFv scFv scFv scFv scFv-Fc scFv scFv scFv scFv scFv
ER, cytoplasm Cytoplasm, ER ER Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm, nucleus Cytoplasm, nucleus Cytoplasm
[28–30,32–34,48–53] [31,48,54,55] [35] [4,19,20,36,56–67] [37,68] [38] [39] [40,65,69] [44] [41,42,70] [43] [45] [47] [46]
Cytoplasm ER ER
R.E. Kontermann / Methods 34 (2004) 163–170
165
Table 2 Intrabodies for the treatment of HIV-1 infections Target
Target function
Antibody format
Subcellular compartment
References
HIV-1 gp120 HIV-1 gp41 CCR5
Viral coat protein Viral coat protein Chemokine receptor, HIV-1 infection HIV-1 co-receptor Reverse transcriptase Virus replication Viral RNA stability Integrase Transcription Matrix protein
scFv, Fab scFv scFv
ER ER, trans-Golgi ER
[72,73,79,80] [74] [81]
scFv scFv, VH–CH1 scFv-C scFv scFv scFv–Vpr scFv Fab, scFv, scFv-C
ER Cytoplasm Cytoplasm, nucleus Cytoplasm Cytoplasm, nucleus, virion Cytoplasm Cytoplasm, nucleus
[82] [83–85] [86–90] [75,91–95] [76,77,96] [78] [97–99]
CXCR4 HIV-1 RT HIV-1 Tat HIV-1 Rev HIV-1 IN Cyclin T1 HIV-1 p17
Table 3 Intrabodies for the treatment of other diseases Target
Target function
Antibody format
Subcellular compartment
References
Flavivirus envelope protein HCV NS3 Huntingtin IL-2R MHC-I 1,3-Galactosyl-transferase Root-knot nematode secretions Colicin A
Virus infectivity Non-structural 3 protein Mutated in Huntington’s disease Cytokine receptor Antigen presentation Glycosylation Nematode infection Bacteriotoxin
scFv scFv scFv scFv scFv scFv scFv scFv
Cytoplasm Cytoplasm Cytoplasm, nucleus ER ER ER ER Bacterial periplasm
[104] [105] [102,103] [106–108] [100] [101] [109] [110]
have been generated by genetic engineering and molecular evolution or by the employment of molecular chaperones [5–14]. Another approach is the direct in vivo selection or screening of stable antibody fragments from intrabody libraries. Such intrabodies are either identiWed by their ability to bind an antigen (intracellular antibody capture technology) ([15–17]; see also Cattaneo et al., this issue) or by an antigen-independent selection using a selectable marker fused to the scFv fragments ([18]; see also Maur et al., this issue). However, a reduced stability of cytoplasmic intrabodies does not necessarily mean that they are non-functional. A recent study of several anti-ras scFv intrabodies showed that expression of instable intrabodies in the cytoplasm leads to antigen-speciWc intracellular coaggregation of scFvs with its antigen. This indicates that the formation of such aggregates might be a general mode of action of cytoplasmic intrabodies for the generation of phenotypic knockouts [19,20].
ity is mediated by the intrabody, e.g., by targeting viral encoded or overexpressed/abnormal proteins, target-cell speciWcity of gene transfer is not essential, although it might inXuence therapeutic eYcacy. Gene transfer can be achieved by a variety of methods. Viral (e.g., retroviruses, adenoviruses, and adeno-associated viruses) and non-viral (e.g., liposomes, molecular conjugates) transfer systems are the most widely studied vehicles for gene transfer. Both systems have advantages and disadvantages associated with eYcacy, target cell speciWcity, non-speciWc uptake, immunogenicity, toxicity, and safety issues [23–25]. Regarding intrabodies, retroviral and adenoviral gene transfer systems have been developed to deliver intrabody genes into target cells ex vivo or in vivo [26,27]. These studies include one phase I trial for the treatment of ovarian cancer patients with an anti-erbB2 intrabody [28]. As described below this study demonstrated the need for highly eYcient gene transfer and/or the combination with strategies leading to bystander eVects in order to achieve a therapeutic beneWt.
4. Transfer of intrabody-encoding genes A key issue in the therapeutic use of intrabodies is the eYcient transfer of the intrabody-encoding gene into target cells. Target cell speciWcity of gene transfer is necessary if the intrabody targets a general pathway found in normal and diseased cells. Target cell speciWcity can be achieved either through transductional or transcriptional targeting [21,22]. On the other hand, if the speciWc-
5. Tumour therapy Antibodies are widely accepted therapeutic agents for the treatment of cancer, which is reXected by the growing number of approved antibodies and antibodies tested in preclinical and clinical studies. These antibodies are selected to act on extracellular targets after systemic
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or local administration. Functionally, these antibodies possess either neutralising activities or induce a humoral or cellular immune response leading to the destruction of tumour cells or the associated tumour vasculature. In addition, eYcacy can be increased by the conjugation of potent cytotoxic components such as drugs, toxins, or radionuclides. Antibodies can also be engaged as intrabodies aVecting intracellular targets and pathways associated with tumour cell proliferation, diVerentiation, and invasion. 5.1. Inhibition of receptor tyrosine kinases Receptor tyrosine kinases (RTKs) are regulators of cell growth and diVerentiation. Binding of a ligand to the extracellular domain results in receptor dimerisation and subsequent phosphorylation of intracellular substrates coupled to signal transduction pathways which regulate gene expression. Members of the epidermal growth factor RTK receptor family, e.g., EGF receptor and erbB2, are overexpressed by a variety of tumours including breast and ovary carcinoma. Intrabodies have been extensively studied as inhibitors of members of the EGF receptor family (see Table 1). Phenotypic knockout of EGFR or erbB2 was achieved by retention of the receptor molecules in the ER employing intrabodies equipped with an ER retention signal. This caused inhibition of tumour cell proliferation in vitro and in vivo and the eradication of the tumour cells by the induction of apoptosis [29–33]. Cytotoxic eVects could be correlated with the expression level of intrabodies and their target protein [34]. Antineoplastic eVects were also observed in animal models after adenovirus-mediated transfer of the intrabody-encoding gene into ovarian cancers [27]. This led to a phase I trial utilising an anti-erbB2 scFv-encoding adenovirus to treat ovarian cancer patients [28]. Although none of the patients treated showed dramatic clinical beneWts, due to gene transfer only into a limited number of tumour cells, this trial demonstrated the feasibility of this approach but highlighted also the need for eYcient gene transfer systems to transduce as many tumour cells as possible. The approach to inhibit the function of receptor tyrosine kinases by intrabodies is also applicable to other receptors than members of the EGF receptor family. Recently, novel intrabodies against the oncogenic protein BCR-ABL have been selected by an intracellular capture technology [35]. 5.2. Intrabodies inhibiting intracellular signal transduction pathways The Ras protein is a guanine nucleotide-binding protein which catalyses the hydrolysis of GTP to GDP. Its biochemical function depends on its GTP-binding and catalytic activity and on the correct cellular location at
the inner surface of the plasma membrane. Ras plays a central role in the signal transduction cascade of receptor tyrosine kinases acting on various targets (PI3kinase, Raf, and Ral). Overexpression or mutations in the ras gene are associated with a transformed phenotype of a variety of tumours leading to disregulation of cell growth and diVerentiation. Raf-1, a serine–threonine kinase, is one of the important down-stream eVectors of Ras function. Complexation of Ras with Raf-1, normally found in the cytoplasm, leads to translocation of Raf-1 to the plasma membrane and subsequent activation of its kinase function. The correct localisation of Ras to the inner surface of the plasma membrane is crucial for its function in stimulation of cellular proliferation. Studies with anti-ras intrabodies have shown that diverting Ras from the inner compartment of the plasma membrane, e.g., by forming cytoplasmic aggresomes, results in inhibition of its function (see Table 1). Furthermore, neutralisation of ras with a scFv intrabody derived from antibody Y13-259 promoted apoptosis in human cancer cells and led to tumour regression of a colon carcinoma tumour model in nude mice [36]. 5.3. The apoptotic pathway as target for intrabodies Programmed cell death (apoptosis) is an important process controlling the equilibrium of loss and growth of cells in an organism and the elimination of damaged or transformed cells. Programmed cell death is regulated by the interplay of pro-apoptotic and anti-apoptotic proteins. Apoptosis is carried out by a family of cysteine proteinases (caspases). A central role in this process plays the executioner caspase 3. Caspase 3 is activated by upstream caspases leading to the formation of an active tetrameric enzyme. It has been shown that forced dimerisation can cause self-activation of capase 3 and the induction of cell death. A scFv-fusion protein consisting of an anti--galactosidase scFv fused to caspase 3 was able to bring two caspase 3 domains in close proximity when co-expressed intracellularly with -galactosidase [37]. This led to autoactivation and triggering of apoptosis. Importantly, activation depended on dimerisation and the scFv–caspase fusion protein was non-toxic in normal cells. For tumour therapy, proteins such as p53 mutants or oncogenic chimeric fusion proteins resulting from chromosomal translocations may be candidate targets for the scFv–caspase 3 approach. Although applicability has not yet been demonstrated in such a setting, the use of intracellular scFv-fusion proteins which can activate an eVector function upon scFv-mediated dimerisation opens up a new way to regulate pathways involved in cell viability or tumourigenicity. Other studies of targeting the apoptotic pathway with intrabodies described the use of anti-caspase 7 as antigen, another proapoptotic proteinase [38]. Furthermore, intrabodies inhibiting the anti-apoptotic protein Bcl-2 enhanced
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drug-mediated cytotoxicity of Bcl-2 overexpressing tumour cells [39]. 5.4. Nuclear targets of intrabodies Intrabodies targeting nuclear targets have been described to induce growth arrest and cell death. These targets include cell cycle regulators, p53, transcription factors, viral and cellular oncogenic proteins, or structural components of the chromatin (see Table 1). Intrabodies which are able to restore the DNA-binding and therefore transcriptional activity of certain p53 mutants have recently been described [40]. These intrabodies are one of the few examples where an antibody is able to revert an inactive mutant back into a functional molecule. Such intrabodies may represent a novel class of molecules for p53-based cancer therapy. Upregulation of transcription factors is often associated with malignancies. The heterodimeric transcription factor ATF-1/CREB, involved in cAMP and Ca2+ induced transcriptional activity, is upregulated in metastatic melanoma cells [41,42]. An intrabody inhibiting binding of ATF-1 to DNA was able to reduce CRE-dependent promoter activity and to suppress tumourigenicity and metastatic potential in a melanoma cancer animal model. The viral oncoprotein E7 of the human papillomavirus type 16 is found in over 90% of cervical cancers and plays a major role in tumourigenicity through interaction with several proteins involved in transcription and cell cycle regulation, e.g., the retinoblastoma protein pRb. An intrabody directed against E7 was able to down-regulate E7 and to inhibit cell proliferation in vitro [43]. Cell cycle regulation has been targeted with an intrabody directed against cyclin E [44]. Cyclin E plays a central role in regulating cell cycle progression from G1 to S phase in normal cells. Dysregulated cyclin E is found in nearly all breast cancers and leads to an accelerated cell cycle and chromosome instability. The anti-cyclin E intrabody was expressed as scFv–Fc fusion protein resulting in a dramatic increase in half-life (see also Strube and Chen; this issue). Targeting of the intrabody to the nucleus caused growth inhibition of a breast cancer cell line. This indicates that inhibiting cell cycle regulation might be a potential target for cancer therapy. Heterochromatin protein 1 (HP1) is involved in silencing gene expression, nuclear envelope re-assembly, recruiting of proteins to the mitotic spindle, and spermatogenesis. An intrabody directed against the chromodomain (CD) of HP1 triggered a p53-independent apoptotic pathway leading to a cell-lethal phenotype in mouse or human Wbroblasts [45]. 5.5. Extracellular proteinases as targets Various proteinases expressed by transformed cells are involved in tumour progression or play a central role
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in tumour invasion and metastasis formation. Cathepsin L, a cysteine proteinase, is found intracellularly in lysosomes where it degrades a wide range of cytoplasmic and nuclear proteins. Cathepsin L is also overexpressed and secreted by tumour cells, e.g., melanoma cells contributing to tumour progression. An intrabody directed against the procathepsin L form was shown to cause inhibition of secretion and intracellular accumulation of the protein [46]. Although inhibition of tumour growth has not been demonstrated, anti-cathepsin L intrabodies may be useful tools to inhibit the tumourigenic and metastatic phenotype of human melanoma cells. Another type of proteinases, the metalloproteinases MMP-2 and MMP-9 which both degrade collagenase IV in the basement membrane, play an important role in tumour invasion and the formation of metastasis. Expression of anti-type IV collagenase intrabody in the ER of a highly metastatic lung carcinoma cell line resulted in down-regulation of collagenase expression and the inhibition of invasion in an in vitro colony formation assay [47]. Such intrabodies directed against tumour-associated proteinases may represent a novel approach for the inhibition of tumour invasion in vivo.
6. Treatment of infectious diseases 6.1. HIV-1 Over 40 million people were infected with the human immunodeWciency virus 1 (HIV-1) at the end of 2002 with a lethality of approximately 7%. HIV-1 mainly infects T cells and macrophages leading to immunodeWciency syndromes. The CD4 receptor and various co-receptors on the target cell surface mediate virus absorption and infection. The virus encodes a total of 15 distinct proteins including structural proteins or proteins involved in virus replication, gene regulation, processing of viral proteins and the genome, and virus assembly [71]. All these proteins are potential targets for intrabodies. Intrabodies directed against several of these proteins including the viral coat proteins gp120, and gp41, the matrix protein p17, proteins involved in transcription and replication (reverse transcriptase, Tat, Rev) or integration of the viral DNA into the host genome (integrase) have been described (see Table 2). These intrabodies eYciently inhibited HIV-1 production in host cells. Intrabodies directed against gp120 were able to inhibit virus production when expressed in the ER. This approach was extended to a combined intra- and extracellular immunisation using the same antibody to block binding of the virus to CD4 [72]. Lymphocytes transduced with the intrabody expression cassette using an adeno-associated virus shuttle vector did not show any cytotoxicity and were resistant to infection by several primary HIV-1 patient isolates [73]. In another study,
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non-neutralising anti-gp41 intrabodies directed to the ER or trans-Golgi network bound to gp160 and inhibited HIV-1 replication. Intrabodies expressed in the ER were able to block processing of gp160 into gp120 and gp41, in contrast to intrabodies localised in the trans-Golgi network, indicating that the intrabodies use diVerent mechanisms to inhibit HIV-1 replication [74]. Importantly, this study showed that non-neutralising antibodies are useful therapeutic agents when expressed intracellularly. Most neutralising antibodies are directed against epitopes which show a high rate of mutation, while epitopes recognised by non-neutralising antibodies are often more conserved among virus isolates. Thus, the use of such non-neutralising intrabodies may represent an advantage for the development of intrabodies to Wght HIV-1. The replication of viral DNA was targeted with cytoplasmic or nuclear intrabodies inhibiting the reverse transcriptase or the transactivators Tat or Rev (see Table 2). The use of Rev as target has several advantages as it is essential for posttranscriptional processing of the initial viral transcript, is speciWc for a highly conserved Rev-responsive element (RRE), and exhibits favourable threshold kinetics [75]. The inhibitory activity of antiRev intrabodies was further improved by combining them with an RRE decoy RNA targeting both the Rev protein and Rev-binding site which resulted in eYcient inhibition of infection by various virus isolates. Another target is the HIV-1 integrase (IN) packaged into the virus particle and involved in virus replication. Cytoplasmic or nuclear expression of anti-IN intrabodies recognising the catalytic or C-terminal domains of IN resulted in resistance to productive HIV-1 infection [76]. In another approach, an anti-integrase scFv was expressed in fusion with the viral accessory protein Vpr which is packaged into the virion in high quantities. The fusion protein was eYciently encapsidated into the virion resulting in a decreased infectivity. Thus, this intra-virion gene therapy approach, which targets virus replication as well as virion spread, leads to increased inhibitory eVects [77]. Targeting of host proteins involved in HIV-1 infection and/or replication represents another approach for HIV-1 therapy as phenotypic knockout of HIV-1 receptors or co-receptors should lead to inhibition of HIV-1 infection. Two coreceptors, CXCR4 and CCR5, have been demonstrated to be relevant for HIV-1 cell entry in vivo. While CCR5 is involved in the primary infection with R5 viruses, disease progression correlates with the evolvement of X4 strains which use CXCR4 as major co-receptor. Both receptors have been targeted with intrabodies expressed in the ER (see Table 2) causing functional deletion or down-regulation of the receptor and resistance to viral infection. Other targets are host proteins necessary for virus replication. One such protein is human cyclin T1 (hCyclinT1) which is a subunit of the positive transcription elongation factor (P-TEFb)
complex. This protein interacts with Tat and binds cooperatively to the transactivation response element (TAR) leading to transactivation of HIV-1 transcription. AntihCyclinT1 intrabodies directed against the cyclin box domain were able to block HIV-1 replication. Importantly, they did not cause cellular toxicity making these intrabodies useful agents for HIV-1 therapy [78]. 6.2. Other infectious diseases Intrabodies have also been described for the treatment of various other viral infections including the inhibition of tick-borne Xavivirus infection with a neutralising intrabody directed against the envelope protein and the inhibition of hepatitis C virus (HVC) replication with an intrabody recognising the non-structural 3 protein (NS3) (see Table 3). Although as of yet only few intrabody approaches have been described for the therapy of bacterial or parasite infections (see Table 3), intrabodies should have the potential to treat these diseases by targeting the entry or replication/assembly pathway or by directly neutralising toxicity of a target protein.
7. Intracellular immune suppression Allocraft rejection is caused by cellular as well as humoural components of the immune system and the inhibition of these events is essential for a successful transplantation. One of the central processes leading to rejection is the recognition of MHC class I antigens of the donor cells by host T cells. In a recent study, antiMHC-I heavy chain intrabodies directed to the ER were described to induce marked down-regulation of MHC-I surface expression in adenovirus transduced primary human keratinocytes, even in the presence of MHC-I upregulating cytokines [100]. MHC-I knockout might Wnd applications in impairing alloantigen expression and prolonging the survival of allografts. Another approach used an intrabody against the enzyme 1,3-galactosyltransferase to inhibit 1,3-galactosylation of cell surface antigens [101]. This carbohydrate structure is a major antigen recognised by xenoreactive natural antibodies in higher primates. The intrabody was able to reduce cytotoxicity mediated by anti-Gal- 1,3Gal antibodies in vitro by more than 90%. Although the enzymatic activity was not completely knocked out, such intrabodies may be useful to protect cells from elicited antibodies or innate eVectors.
8. Other applications Intrabodies may also Wnd applications for the treatment of diseases associated with the expression of altered proteins responsible for a pathogenic phenotype which
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are normally not found in healthy cells. One such example is the Huntington’s disease (HD), a neurodegenerative disorder caused by extended polyglutamine stretches of the huntingtin protein. Stretches of more than 40 glutamines are lethal in subpopulations of neurons in the striatum and cortex, which is associated with the formation of insoluble aggregates in aVected neurons. The in vitro analysis of various intrabodies recognising diVerent regions of the huntingtin protein showed that intrabodies directed against a polyproline stretch or the N-terminus are able to inhibit aggregation as well as cell death while anti-polyglutamine intrabodies stimulated aggregation and apoptosis [102,103]. Such inhibitory intrabodies may be of therapeutic value in the treatment of HD.
9. Concluding remarks Although far from being clinically approved therapeutic agents, intrabodies extend the application of antibodies by targeting antigens not accessible by circulating antibody molecules. The development of intracellularly expressed antibodies introduced a novel way of antigen neutralisation by diverting it from its normal subcellular location causing a phenotypic knockout. As summarised in this article, a large number of diVerent applications have already been established for intrabodies. Certainly, in the future intrabodies will be developed against additional targets and employed for the treatment of a variety of other diseases. However, there remain problems to be solved including improved expression and stability of intrabodies and the development of eYcient and safe gene transfer systems.
References [1] S. Biocca, A. Cardinale, A. Cataneo, in: R.E. Kontermann, S. Dübel (Eds.), Antibody Engineering, Springer Heidelberg Press, New York, 2001, pp. 755–774. [2] P.J. Hudson, Curr. Opin. Biotechnol. 9 (1998) 395–402. [3] R.E. Kontermann, R. Müller, J. Immunol. Methods 226 (1999) 179–188. [4] S. Biocca, F. Ruberti, M. Tafani, P. Pierandrei-Amaldi, A. Cattaneo, Bio/Technology 13 (1995) 1110–1115. [5] P. Jurado, d. Ritz, J. Beckwith, V. de Lorenzo, L.A. Fernandez, J. Mol. Biol. 320 (2002) 1–10. [6] J.C. Laden, P. Philibert, F. Torreilles, M. Pugniere, P. Martineau, Res. Microbiol. 153 (2002) 469–474. [7] P. Martineau, J.M. Betton, J. Mol. Biol. 292 (1999) 921–929. [8] P. Martineau, P. Jones, G. Winter, J. Mol. Biol. 280 (1998) 117–127. [9] E.C. Ohage, B. Steipe, J. Mol. Biol. 291 (1999) 1119–1128. [10] P. Writz, B. Steipe, Protein Sci. 8 (1999) 2245–2250. [11] E.C. Ohage, P. Writz, J. Barnikow, B. Steipe, J. Mol. Biol. 291 (1999) 1129–1134. [12] A. Wörn, A. Auf der Maur, D. Escher, A. Honegger, A. Barberis, A. Plückthun, J. Biol. Chem. 275 (2000) 2795–2803. [13] K. Proba, A. Wörn, A. Honegger, A. Plückthun, J. Mol. Biol. 275 (1998) 245–253. [14] H. Bach, Y. Mazor, S. Shaky, A. Shoham-Lev, Y. Berdichevsky, D.L. Gutnick, I. Benhar, J. Mol. Biol. 312 (2001) 79–93.
169
[15] M. Visintin, E. Tse, H. Axelson, T.H. Rabbitts, A. Cattaneo, Proc. Natl. Acad. Sci. USA 96 (1999) 11723–11728. [16] T. Tanaka, G.T. Chung, A. Forster, M.N. Lobato, T.H. Rabbitts, Nucleic Acids Res. 31 (2003) e23. [17] A. auf der Maur, C. Zahnd, F. Fischer, S. Spinelli, A. Honegger, C. Cambillau, D. Escher, A. Plückthun, A. Barberis, J. Biol. Chem. 277 (2002) 45075–45085. [18] A. auf der Maur, D. Escher, A. Barberis, FEBS Lett. 508 (2001) 407–412. [19] M. Lener, I.R. Horn, A. Cardinale, S. Mesina, U.B. Nielsen, S.M. Rybak, H.R. Hoogenboom, A. Cattaneo, S. Biocca, Eur. J. Biochem. 267 (2000) 1196–1205. [20] A. Cardinale, I. Filesi, S. Biocca, Eur. J. Biochem. 268 (2001) 268– 277. [21] K.-W. Peng, S.J. Russell, Curr. Opin. Biotechnol. 10 (1999) 454– 457. [22] D.V. SchaVer, D.A. LauVenburger, Curr. Opin. Mol. Ther. 2 (2000) 155–161. [23] I. Verma, N. Somia, Nature 389 (1997) 239–242. [24] F. Meyer, M. Finer, Cell. Mol. Biol. 47 (2001) 1277–1294. [25] A. Mountain, Trends Biotechnol. 18 (2000) 119–128. [26] D. Zhan, A. Murakami, R.P. Johnson, J. Sui, J. Cheng, J. Bai, W.A. Marasco, J. Acquir. Immune DeWc. Syndr. 32 (2003) 245– 254. [27] J. Deshane, G.P. Siegal, M. Wang, M. Wright, R.P. Bucy, R.D. Alvarez, D.T. Curiel, Gynecol. Oncol. 64 (1997) 378–385. [28] R.D. Alvarez, M.N. Barnes, J. Gomez-Navarro, M. Wang, T.V. Strong, W. Arafat, R.B. Arani, M.R. Johnson, B.L. Roberts, G.P. Siegal, D.T. Curiel, Clin. Cancer Res. 6 (2000) 3081–3087. [29] R.R. Beerli, W. Wels, N.E. Hynes, J. Biol. Chem. 269 (1994) 23931–23936. [30] J. Deshane, F. Loechel, R.M. Conry, G.P. Siegal, C.R. King, D.T. Curiel, Gene Ther. 1 (1994) 332–337. [31] C.B. Jannot, R.R. Beerli, S. Mason, W.J. Gullick, N.E. Hynes, Oncogene 13 (1996) 275–282. [32] J. Deshane, G. Cabrera, J.E. Grim, G.P. Siegal, J. Pike, R.D. Alvarez, D.T. Curiel, Gynecol. Oncol. 59 (1995) 8–14. [33] J. Deshane, J . Grim, S. Loechel, G.P. Siegal, R.D. Alvarez, D.T. Curiel, Cancer Gene Ther. 3 (1996) 89–98. [34] J. Grim, J. Deshane, G.P. Siegal, R.D. Alvarez, P. DiFiore, D.T. Curiel, J. Mol. Med. 76 (1998) 451–458. [35] E. Tse, M.N. Lobato, A. Forster, T. Tanaka, G.T. Chung, T.H. Rabbitts, J. Mol. Biol. 317 (2002) 85–94. [36] O. Cochet, M. Kenigsberg, I. Delumeau, A. Vvirone-Oddos, M.C. Multon, W.H. Fridman, F. SchweighoVer, J.L. Teillaud, B. Tocques, Cancer Res. 58 (1998) 1170–1176. [37] E. Tse, T.H. Rabbitts, Proc. Natl. Acad. Sci. USA 97 (2000) 12266–12271. [38] Q. Zhu, C. Zeng, A. Huhalov, J. Yao, T.G. Turi, D. Danley, T. Hynes, Y. Cong, D. DiMattia, S. Kennedy, G. Daumy, E. SchaeVer, W.A. Marasco, J.S. Huston, J. Immunol. Methods 231 (1999) 207–222. [39] A. Piche, J. Grim, C. Rancourt, J. Gomez-Navarro, J.C. Reed, D.T. Curiel, Cancer Res. 58 (1998) 2134–2140. [40] C. de Fromentel, N. Gruel, C. Venot, L. Debussche, E. Conseiller, C. Dureuil, J.L. Teillaud, B. Tocque, L. Bracco, Oncogene 18 (1999) 551–557. [41] D. Jean, M. Bar-Eli, Crit. Rev. Immunol. 21 (2001) 275–286. [42] D. Jean, C. Tellez, S. Huang, D.W. Davis, C.J. Bruns, D.J. McConkey, S.H. Hinrichs, M. Bar-Eli, Oncogene 19 (2000) 2721– 2730. [43] F. Wang-Johanning, G.Y. Gillespie, J. Grim, C. Rancourt, R.D. Alvarez, G.P. Siegal, D.T. Curiel, Cancer Res. 58 (1998) 1893–1900. [44] R.W. Strube, S.-Y. Chen, J. Immunol. Methods 263 (2002) 149– 167. [45] I. Filesi, A. Cardinale, S. van der Sar, I.G. Cowell, P.B. Singh, S. Biocca, J. Cell Sci. 115 (2002) 1803–1813.
170
R.E. Kontermann / Methods 34 (2004) 163–170
[46] N. Guillaume-Rousselet, D. Jean, R. Frade, Biochem. J. 367 (2002) 219–227. [47] W. Wang, J. Zhou, L. Xu, Y. Zhen, J. Environ. Pathol. Toxicol. Oncol. 19 (2000) 61–68. [48] R.R. Beerli, W. Wels, N.E. Hynes, Breast Cancer Res. Treat. 38 (1996) 11–17. [49] J. Deshane, G.P. Siegal, R.D. Alvarez, M.H. Wang, M. Feng, M. Feng, G. Cabrera, T. Liu, M. Kay, D.T. Curiel, J. Clin. Invest. 96 (1995) 2980–2989. [50] D. Graus-Porta, R.R. Beerli, N.E. Hynes, Mol. Cell. Biol. 15 (1995) 1182–1191. [51] M. Wright, J. Grim, J. Deshane, M. Kim, T.V. Strong, G.P. Siegal, D.T. Curiel, Gene Ther. 4 (1997) 317–322. [52] J. Grim, J. Deshane, M. Feng, A. Lieber, M. Kay, D.T. Curiel, Am. J. Respir. Cell. Mol. Biol. 15 (1996) 348–354. [53] W. Arafat, J. Gomez-Navarro, J. Xiang, G.P. Siegal, R.D. Alvarez, D.T. Curiel, Cancer Gene Ther. 7 (2000) 1250–1256. [54] R.R. Beerli, W. Wels, N.E. Hynes, Biochem. Biophys. Res. Commun. 204 (1994) 666–672. [55] S. Hyland, R.R. Beerli, C.F. Barbas III, N.E. Hynes, W. Wels, Oncogene 22 (2003) 1557–1567. [56] S. Biocca, M.S. Neuberger, A. Cattaneo, EMBO J. 9 (1990) 101–108. [57] S. Biocca, A. Di Luzio, T. Werge, A. Cattaneo, Cytotechnology 5 (Suppl. 1) (1991) 49–50. [58] S. Biocca, P. Perandrei-Amaldi, A. Cattaneo, Biochem. Biophys. Res. Commun. 197 (1993) 422–427. [59] S. Biocca, P. Perandrei-Amaldi, N. Campioni, A. Cattaneo, Bio/ Technology 12 (1993) 396–399. [60] A. Cardinale, M. Lener, S. Messina, A. Cattaneo, S. Biocca, FEBS Lett. 439 (1998) 197–202. [61] T.M. Werge, S. Biocca, A. Cattaneo, FEBS Lett. 274 (1990) 193–198. [62] T.M. Werge, C.T. Baldari, J.L. Telford, FEBS Lett. 351 (1994) 393–396. [63] X. Montano, A. Jimenez, Cell Growth DiV. 6 (1995) 597–605. [64] O. Cochet, M. Kenigsberg, I. Delumeau, M. Duchesne, F. SchweighoVer, B. Tocque, J.L. Teillaud, Mol. Immunol. 35 (1998) 1097–1110. [65] O. Cochet, N. Gruel, W.H. Frisman, J.L. Teillaud, Cancer Detect. Prev. 23 (1999) 506–510. [66] T. Tanaka, T.H. Rabbitts, EMBO J. 22 (2003) 1025–1035. [67] E. Tse, G. Chung, T.H. Rabbitts, Methods Mol. Biol. 185 (2002) 433–446. [68] A. Rajpal, T.G. Turi, J. Biol. Chem. 276 (2001) 33139–33146. [69] P.A. Cohen, J.C. Mani, D.P. Lane, Oncogene 17 (1998) 2445–2456. [70] J.M. Bosilevac, C.A. Gilchrist, P.E. Jankowski, S. Paul, A.R. Rees, S.H. Hinrichs, J. Biol. Chem. 273 (1998) 16874–16879. [71] A.D. Frankel, J.A. Young, Annu. Rev. Biochem. 67 (1998) 1–25. [72] S.-Y. Chen, Y. Khouri, J. Bagley, W.A. Marasco, Proc. Natl. Acad. Sci. USA 91 (1994) 5932–5936. [73] J.D. Chen, Q. Yang, A.G. Yang, W.A. Marasco, S.-Y. Chen, Hum. Gene Ther. 7 (1996) 1515–1525. [74] P. Zhou, S. Goldstein, K. Devadas, D. Tewari, A.L. Notkins, J. Immunol. 160 (1998) 14889–14896. [75] R.T. Inouye, B. Du, D. Boldt-Houle, A. Ferrante, I.-W. Park, S.M. Hammer, L. Duan, J.E. Groopman, R.J. Pomerantz, E.F. Terwilliger, J. Virol. 71 (1997) 4071–4078. [76] P. Levy-Mintz, L. Duan, H. Zhang, B. Hu, G. Dornadula, M. Zhu, J. Kulkosky, D. Bizub-Bender, A.M. Skalka, R.J. Pomerantz, J. Virol. 70 (1996) 8821–8832. [77] M. BouHamdan, J. Kulkosky, L.X. Duan, R.J. Pomerantz, J. Hum. Virol. 3 (2000) 6–15. [78] J. Bai, J. Sui, R.Y. Zhu, A.S. Tallarico, F. Gennari, D. Zhang, W.A. Marasco, J. Biol. Chem. 278 (2003) 1433–1442. [79] W.A. Marasco, W.A. Haseltine, S.-Y. Chen, Proc. Natl. Acad. Sci. USA 90 (1993) 7889–7893. [80] S.-Y. Chen, J. Bagley, W.A. Marasco, Hum. Gene Ther. 5 (1994) 595–601.
[81] P. Steinberger, J . Andris-Widhopf, B . Buhler, B.E. Torbett, C.F. Barbas III, Proc. Natl. Acad. Sci. USA 97 (2000) 805–810. [82] M. BouHamdan, D.S. Strayer, D. Wei, M. Mukhtar, L.X. Duan, J. Hoxie, R.J. Pomerantz, Gene Ther. 8 (2001) 408–418. [83] F. Shaheen, L. Duan, M. Zhu, O. Bagasra, R.J. Pomerantz, J. Virol. 70 (1996) 3382–4000. [84] N. Gargano, S. Biocca, A. Bradbury, A. Cattaneo, J. Virol. 70 (1996) 7706–7712. [85] N. Gargano, A. Cattaneo, J. Gen. Virol. 78 (1997) 2591–2599. [86] A.M. Mhashilkar, J. Bagley, S.-Y. Chen, A.M. Szilvay, D.G. Helland, W.A. Marasco, EMBO J. 14 (1995) 1542–1551. [87] A.M. Mhashilkar, D.K. Biswas, J . LaVecchio, A.B. Pardee, W.A. Marasco, J. Virol. 71 (1997) 6486–6494. [88] A.M. Mhashilkar, J. LaVecchio, B. Eberhardt, J. Porter-Brooks, S. Boisot, J.H . Dove, C. Pumphrey, X. Li, R.N. Weissmahr, D.B. Ring, U. Ramstedt, W.A. Marasco, Hum. Gene Ther. 10 (1999) 1453–1467. [89] W.A. Marasco, J. LaVecchio, A. Winkler, J. Immunol. Methods 231 (1999) 223–238. [90] M.C. Poznansky, J. La Vecchio, S. Silva-Arietta, J. PorterBrooks, K. Brody, I.T. Olszak, G.B. Adams, U. Ramstedt, W.A. Marasco, D.T. Scadden, Hum. Gene Ther. 10 (1999) 2505–2514. [91] L. Duan, O. Bagasra, M.A. Laughlin, J.W. Oakes, R.J. Pomerantz, Proc. Natl. Acad. Sci. USA 91 (1994) 5057–5079. [92] L.X. Duan, H. Zhang, J.W. Oakes, O. Bagasra, R.J. Pomerantz, Hum. Gene Ther. 5 (1994) 1315–1324. [93] L.X. Duan, M. Zhu, O. Bagasra, R.J. Pomerantz, Hum. Gene Ther. 6 (1995) 1561–1571. [94] L. Duan, M. Zhu, I. Ozaki, H. Zhang, D.L. Wei, R.J. Pomerantz, Gene Ther. 4 (1997) 533–543. [95] Y. Wu, L. Duan, M. Zhu, B. Hu, S. Kubota, O. Bagasra, R.J. Pomerantz, J. Virol. 70 (1996) 3290–3297. [96] M. BouHamdan, L.X. Duan, R.J. Pomerantz, D.S. Strayer, Gene Ther 6 (1999) 660–666. [97] R. Levin, A.M. Mhashilkar, T. Dorfman, A. Bukovsky, C. Zani, J. Bagley, J. Hinkula, M. Niedrig, J. Albert, B. Wahren, H.G. Gottlinger, W.A. Marasco, Mol. Med. 3 (1997) 96–110. [98] D. Tewari, S.L. Goldstein, A.L. Notkins, P. Zhou, J. Immunol. 161 (1998) 2642–2647. [99] D. Tewari, A.L. Notkins, P. Zhou, J. Gene Med. 5 (2003) 182– 189. [100] A.M. Mhashilkar, C. Doebis, M. Seifert, A. Busch, C. Zani, J. Soo Hoo, M. Nagy, T. Ritter, H.D. Volk, W.A. Marasco, Gene Ther. 9 (2002) 307–319. [101] B. Vanhove, B. Charreau, A. Cassard, C. Pourcel, J.P. Soulilou, Transplantation 66 (1998) 1477–1485. [102] A. Khoshnan, J. Ko, P.H. Patterson, Proc. Natl. Acad. Sci. USA 99 (2002) 1002–1007. [103] J.M. Lecerf, T.L. Shirley, Q. Zhu, A. Kazantsev, P. Amersdorfer, D.E. Housman, A. Messer, J.S. Huston, Proc. Natl. Acad. Sci. USA 98 (2001) 4764–4769. [104] W. Jiang, K. Venugopal, E.A. Gould, J. Virol. 69 (1995) 1044– 1049. [105] D.E. Sullivan, M.U. Mondelli, D.T. Curiel, V. Krasnykh, G. Mikheeva, P. Gaglio, C.B. Morris, S. Dash, M.A. Gerber, J. Hepatol. 37 (2002) 660–668. [106] J.H. Richardson, J.G. Sodroski, T.A. Waldmann, W.A. Marasco, Proc. Natl. Acad. Sci. USA 92 (1995) 3137–3141. [107] J.H. Richardson, T.A. Waldmann, J.G. Sodroski, W.A. Marasco, Virology 237 (1997) 209–216. [108] J.H. Richardson, W. Hofmann, J.G. Sodroski, W.A. Marasco, Gene Ther. 5 (1998) 635–644. [109] M.N. Rosso, A. Schouten, J. Roosien, T. Borst-Vrenssen, R.S. Hussey, F.J. Gommers, J. Bakker, A. Schots, P. Abad, Biochem. Biophys. Res. Commun. 220 (1996) 255–263. [110] P. Chames, J. Fieschi, D. Baty, D. Duche, J. Bacteriol. 180 (1998) 514–518.