key: cord-0037670-wo42j0ps
authors: Nettelbeck, Dirk M.
title: Bispecific Antibodies and Gene Therapy
date: 2011-07-01
journal: Bispecific Antibodies
DOI: 10.1007/978-3-642-20910-9_18
sha: dfc739ec907d914c19fe56cedfae687164fdb402
doc_id: 37670
cord_uid: wo42j0ps

Gene therapy is the transfer of therapeutic genes, via gene transfer vectors, into patients for therapeutic purposes. Different gene therapy strategies are being pursued, including long-term gene correction of monogenetic diseases, eradication of tumor cells in cancer patients, or genetic vaccination for infectious diseases. Bispecific antibodies and gene therapy are connected in two ways. First, bispecific antibodies are tools of interest for the development of targeted gene transfer vectors. Different gene therapy strategies require different vectors, frequently replication-ablated viruses. Similar to the role of antibody engineering in antibody therapy, the engineering of gene transfer vectors has become key to the implementation of genetic therapies. Cytoablative cancer gene therapy and efficient genetic vaccination, for example, depend on vectors that are targeted to cancer cells and antigen-presenting cells, respectively, in order to avoid side effects and vector sequestration. To this end, bispecific antibodies have been engineered as adapters that link the vector to a specific molecule on the targeted cell and at the same time block the interaction with the native virus receptor. Different formats of bispecific antibodies and related molecules have been developed and succeeded in re-directing vectors to target cells in vitro and in vivo. These adapters also improved gene therapies in animal models. Second, gene transfer is a promising tool for delivery of bispecific antibodies to patients. Therefore, vectors can be injected directly into patients for antibody gene transfer, or cells isolated from patients can be genetically modified in vitro and then re-injected for in vivo antibody production. Genetic antibody delivery, compared with standard antibody injection, can be advantageous with respect to achieving persistent antibody titers or effective antibody biodistribution in patients. Initial studies have shown antibody production and therapeutic activity in animal models, setting the stage for more widespread investigations. Moreover, gene therapy can enable novel therapeutic applications for bispecific antibodies by facilitating the delivery of membrane associated or intracellular antibody formats.

Gene therapy is the transfer of genes into patients' cells for therapeutic purposes ( Fig. 18.1 ). Gene therapy was originally envisioned as a cure for inherited (monogenetic) diseases by gene correction, i.e., by replacing or complementing the causative mutated gene with a functional copy. In recent decades, however, gene therapy has been intensively investigated for treatment of many diseases by transfer of diverse classes of therapeutic genes from various species (Table 18 .1). Examples are genes encoding pathogen antigens for prevention or treatment of infectious diseases (genetic vaccination); genes encoding agonists or antagonists of vascular growth factors for treatment of cardiovascular diseases; or genes that directly or indirectly mediate tumor cell killing for cancer treatment. Gene therapy drugs consist of the therapeutic gene, which defines the mode of therapeutic action, and the gene transfer vector, which needs to facilitate appropriate stability, delivery, and expression of the therapeutic gene ( Fig. 18 .1). Indeed, major efforts in gene therapy research focus on vector development, since the delivery of therapeutic genes is complex and critically determines treatment efficacy. Since the 1990s a multitude of gene therapy clinical trials have been performed with thousands of patients and therapeutic efficacy was demonstrated recently. Examples are the restoration of immunity in SCID patients, restoration of some degree of vision in childhood blindness or inhibition of neurodegeneration (Kohn 2010; Roy et al. 2010; Cartier et al. 2009 ). However, most gene therapy approaches necessitate improved efficacy or selectivity of gene transfer in order to facilitate successful applications in patients.

To ensure proper expression of the therapeutic gene in the patients' cells, a gene therapy vector contains a promoter and a transcription termination/polyadenylation signal ( Fig. 18.1 ). Further regulatory elements can be exploited, for example, to achieve enhanced (introns) or bicistronic (internal ribosome entry sites, IRES) gene expression. Importantly, regulatory elements can be exploited for spatial or temporal control of gene expression. Examples are inducible or cell type-specific promoters or sequences differentially regulating mRNA stability or translation efficiency (Goverdhana et al. 2005; Dorer and Nettelbeck 2009; Brown and Naldini 2009) . To improve stability of the therapeutic DNA, these eukaryotic expression For gene therapy, a therapeutic gene is delivered into the patient's cell, where the gene product is expressed and mediates therapeutic activity. Examples are the complementation of the patient's genetic defects or the killing of cancer cells. For delivery and expression, the therapeutic gene is incorporated into a gene transfer vector containing regulatory sequences (e.g., for transcription start and termination). Frequently, the vector is also a means for efficient gene delivery into the patient's cells (e.g., replication-deficient viruses). Right panels: Bispecific antibodies can be either a tool for targeting gene transfer vectors to specific cell types (1) or gene transfer can be exploited as a tool for antibody therapy by antibody gene transfer and subsequent synthesis of the antibody in the patient (2) Singer and Verma (2008) cassettes are inserted either into circular plasmids, which might be further packaged by non-viral vectors, or into genomes of replication-deficient viral vectors ( into hematopoietic stem cells for treatment of inherited immunodeficiencies (Kohn 2010) . In contrast, transient gene transfer is usually sufficient for genetic vaccination or cytoablative cancer therapy. For the latter, however, efficient gene transfer is pivotal and thus vector choice is determined by transduction efficiency. In this regard, conditionally replication-competent viral vectors have been recently engineered allowing for vector spread in tumors and thus amplified gene transfer (Parato et al. 2005; Cody and Douglas 2009 ). Such replication-competent vectors also mediate tumor cell lysis by virus replication, termed oncolysis or virotherapy. Hence, from the perspective of the virotherapist, insertion of therapeutic genes into the genome of oncolytic viruses is a strategy to complement oncolysis with gene therapy ("arming" of oncolytic viruses).

Many gene therapy applications require the restriction of gene transfer to specific cells. This is obvious for cytoablative gene therapy and for replication-competent vectors. Also effective genetic vaccination can depend on gene transfer into appropriate immune cells, as antigen expression in the wrong cells can trigger tolerance rather than immunity. Consequently, vector targeting is a major challenge for gene therapy research. Targeted gene therapy (or viral replication) can be achieved by inserting cell-binding ligands into the gene transfer vector for targeted cell entry, or by post-entry regulation of therapeutic gene expression using appropriate regulatory sequences, as mentioned above.

Bispecific antibodies and gene therapy are connected in two ways. First, bispecific antibodies have been developed as promising tools for targeting cell entry of gene transfer vectors: as adapter molecules they link the vector to a marker molecule (specifically) expressed on the target cell surface ( Fig. 18.1) . Second, gene therapy can be an alternative means for delivery of therapeutic antibodies to patients, i.e., by antibody production in the patients' cells (genetic antibody therapy, Fig. 18 .1). Besides genetic delivery of (established) soluble antibodies, such antibody gene transfer can also facilitate new applications for (bispecific) antibody therapy, for example by expression of membrane-bound or intracellular derivatives. Certainly combination therapies of bispecific antibodies and gene therapy can also be envisioned.

Bispecific antibodies have been exploited in gene therapy as tools to direct viral gene transfer vectors to diseased cells. Therefore, an antibody with specificity for a viral surface protein is linked to a second antibody that binds to a cell surface molecule of interest, thus implementing an adapter molecule that binds the gene transfer vector to the target cell (Figs. 18.1 and 18.2). Such modification of virus tropism is required when virus receptor expression is lacking on target cells, preventing gene transfer, or when widespread expression of the native virus receptor on healthy cells leads to adverse side effects and vector sequestration. For the latter, either the viral attachment proteins have been mutated without losing their affinity for the adapter, or the receptor-binding domain of the virus attachment protein is shielded by the adapter. The resulting loss of virus tropism for healthy cells is termed de-targeting. Binding of and entry into target cells in both cases is mediated by the target of the cell-binding moiety of the adapter (re-targeting). Important advantages of the adapter strategy are (a) it does not require modifications to the virus structure, which might well turn out to be detrimental for Bispecific antibodies as tools for targeting gene therapy. Bispecific adapters binding to the gene transfer vector via one specificity and to a cell surface molecule with the other are used for delivering therapeutic genes to specific cell types. This strategy is of interest to gene therapy in order to ensure targeted therapy and avoid side effects. These bispecific adapters might contain one antibody or antibody fragment (for either vector or cell binding). Alternatively, they can be bispecific antibodies: chemical conjugate, diabody or tandem scFv vector assembly, stability, or activity; (b) it is flexible as vector binding to any target molecule, to which an antibody can be raised, is possible by exchange of the adapter's cell-binding moiety and (c) once an effective adapter for a specific vector has been generated, it can be used for transfer of any therapeutic gene by corresponding derivatives of this vector.

Bispecific antibodies as adapters for targeting gene transfer have been most intensively investigated with adenoviral vectors. Adenoviruses (Ads, McConnell and Imperiale 2004) possess a double-stranded linear DNA genome covered by a protein capsid, but not a lipid envelope. The receptor-binding spike of the adenoviral capsid, made of the trimeric fiber protein, is responsible for attachment to host cells by binding to the virus receptor, which is the coxsackie-adenovirus receptor (CAR) for the mostly used human Ad serotype 5 (HAdV-5). Virus internalization into the host cell is then mediated by a secondary interaction of a different virus capsid protein, the penton base, with cellular integrins. By separating cell binding from entry, this two-step mechanism facilitates a high degree of flexibility for the nature of initial attachment of Ad vectors to cells. After entry of the vector into the cell, the virus genome is transferred to the nucleus, where viral genes are expressed from the episomal genome. Likewise, therapeutic genes are expressed after transfer of Ad vector genomes into patients' cells. Therefore, essential viral genes are replaced with the therapeutic gene, rendering the vector replication-deficient. More recently, therapeutic genes have been inserted into replication-competent Ads (McConnell and Imperiale 2004). Ads represent prominent gene therapy vectors, as they are stable, can be produced at high titers, possess an effective gene transfer machinery, and are only mildly pathogenic (HAdV-5 causes common cold symptoms) (McConnell and Imperiale 2004). They have been the most frequently used viral vectors in clinical gene therapy trials (Journal of Gene Medicine Clinical Trials Database). These trials have revealed a favorable safety profile of Ad vectors in patients. Cancer gene therapy and genetic vaccination are the regimens where Ad vectors are widely used. One therapeutic approach in cancer gene therapy is molecular chemotherapy, also termed gene-dependent enzyme prodrug therapy (GDEPT). This strategy is based on transfer of a gene encoding a prodrug-activating enzyme, which activates a harmless prodrug into an effective chemotherapeutic drug (Portsmouth et al. 2007 ). The rationale for this strategy is that tumor-restricted prodrug activation should facilitate effective concentrations of the chemotherapeutic drug in the tumor, which cannot be achieved by conventional systemic infusion of the drug due to dose-limiting side effects. GDEPT and other cytoablative cancer gene therapies depend on tumor-selective gene transfer which is not provided by unmodified HAdV-5 or other Ad serotypes due to widespread expression of Ad receptors also on healthy cells. Ads are also frequently used as vectors for genetic vaccination, which is most efficient when the antigen gene is transduced into professional antigen-presenting cells (APCs), which provide the proper signals for activation of immune effector cells. Dendritic cells (DCs) are the most effective APCs, but are difficult to transduce. Though Ads are the most effective gene transfer vectors for DCs, high vector titers are required for efficient DC transduction because of low expression of CAR.

Antibodies are attractive binding molecules for targeting gene transfer vectors based on their high affinity, specificity, and the opportunity to generate antibodies with specificity for virtually any cell surface target molecule. Three strategies have been pursued for insertion of targeting ligands into viral gene transfer vectors: genetic fusion to viral capsid or envelope proteins, complexing with bispecific adapters, or chemical linkage. Major drawbacks of the genetic and chemical strategies are that they are tedious and often interfere with viral functions. Moreover, genetic insertion of antibodies into the adenoviral capsid is hampered by the incompatibility of biosynthesis of capsid and antibody molecules. Ad capsid proteins are synthesized in the cytosol and transferred to the nucleus where viral particle assembly takes place, whereas antibodies are produced via the secretory pathway, which ensures their proper folding. Consequently, genetic fusion of antibodies to Ad capsid proteins has been inefficient and limited to a few cytosolically stable antibody fragments (Hedley et al. 2006; Vellinga et al. 2007; Poulin et al. 2010) . In contrast, synthesis of adapter molecules can be separated from virus production. Moreover, the insertion of cell-binding antibodies into adapter molecules is less tedious than the engineering of a complete new virus genome and resulting adapters can be linked to any Ad vector, allowing for better flexibility. For production of adapters, antibody fragments binding to Ad capsid proteins, mostly the fiber, and antibodies or antibody fragments binding to cell surface target molecules of interest have been used (Fig. 18 .2). They have been linked by chemical conjugation (see also: Chap. 3) or by genetic fusion, the latter generating tandem scFvs or scDbs (see also: Chap. 5). As an alternative to bispecific antibodies, adapters have been generated by linking virus-binding antibody fragments to cell-binding proteins or peptides, or by linking cell-binding antibody fragments to the soluble adenovirus receptor CAR.

The adapter strategy for targeting cell entry of Ad vectors has been pioneered by Douglas and co-workers for targeting of folate receptor overexpressing tumor cells (Douglas et al. 1996) . To this end, they chemically conjugated folate to the Fab fragment of a neutralizing anti-Ad fiber monoclonal antibody (MAb). A Fab fragment was used to avoid agglutination of Ad vectors by bivalent antibodies. After complexation to the respective Ad vector, the adapter mediated folatedependent transfer of a reporter gene or of cytoablative genetic prodrug activation to target cells. The Fab fragment alone inhibited adenoviral transduction, which was expected as it was derived from a neutralizing antibody. Thus the Fab-folate adapter realized targeted gene transfer by both ablating virus binding to the native virus receptor and directing virus attachment to a novel cell surface molecule (Fig. 18.1) . Wickham et al. described a bispecific antibody for directing Ad cell binding to integrins. This adapter consisted of the integrin-binding Mab chemically linked to a second MAb with specificity for a peptide tag, which was engineered into the Ad penton base (Wickham et al. 1996) . The conjugate mediated enhanced adenoviral transduction of human smooth muscle and endothelial cells, which were only modestly tranduced by unmodified HAdV5 vectors. Subsequently, various bispecific antibody conjugates were reported, that consist of a fiber-binding Fab fragment covalently linked to a cell-binding antibody or antibody fragment. Such bispecific antibody conjugates have been reported to re-direct Ad gene transfer to various cell types via binding to different cell surface molecules, including CD40, EpCAM, Tag72, CD70, and ACE (Tillman et al. , 2000 De Gruijl et al. 2002; Brandao et al. 2003; Miller et al. 1998; Haisma et al. 1999; Israel et al. 2001; Reynolds et al. 2000 Reynolds et al. , 2001 . These reports confirm the high flexibility of the adapter approach. For example, DCs, as professional antigen-presenting cells, represent targets of interest for gene transfer aiming at genetic vaccination for infectious or malignant diseases. Conjugates of a-fiber Fab and MAbs binding to the DC surface molecule CD40 allowed for efficient Ad gene transfer into mouse and human DCs (Tillman et al. , 2000 . With this adapter, improved efficiency and selectivity of Ad gene transfer to DCs was also achieved in situ using human skin explants (De Gruijl et al. 2002) . In addition to targeting Ad entry, the a-fiber Fab/aCD40 mAb adapter triggered DC activation, as required for efficient induction of immune responses, via its CD40-binding activity. Accordingly, the adapter increased the efficiency of tumor vaccination with Ad vector transduced DCs in an animal model (Tillman et al. 2000) . Adapter targeting of Ad vectors to cancer cells was demonstrated in cell culture studies with an EGFR-binding a-fiber Fab/MAb conjugate for squamous cell carcinoma, glioblastoma, and osteosarcoma (Miller et al. 1998; Blackwell et al. 1999 ; Barnett et al. 2002) ; with an EpCAM-binding Fab/Fab conjugate for various adenocarcinomas (Haisma et al. 1999; Heideman et al. 2001) ; with a TAG-72-binding a-fiber Fab/MAb conjugate for ovarian cancer (Kelly et al. 2000) ; and with a CD70-binding a-fiber Fab/MAb conjugate for B cell lymphomas (Israel et al. 2001) . As CAR-expression varies on cancer cells, adapters frequently mediated markedly enhanced transduction of cancer cells. Yet another type of antibody-based adapter conjugate has been generated by linking a-fiber Fab fragments to basic fibroblast growth factor for targeting of various cancer cells (FGF2, Goldman et al. 1997; Rogers et al. 1997; Rancourt et al. 1998 ) to a synthetic lung-homing peptide (Trepel et al. 2000) , or to the Hc-fragment of tetanus toxin for targeting neuronal cells (Schneider et al. 2000) .

Recombinant bispecific adapter molecules possess attributes that are advantageous for application in vector targeting when compared with chemical conjugates. Foremost, they can be produced by standardized procedures of prokaryotic or eukaryotic expression yielding well-defined molecules. Both tandem single chain variable fragments (tandem scFvs, see also: Chap. 5) and single chain diabodies (scDbs, see also: Chap. 5) have been used for targeting Ad gene transfer.

Haisma and co-workers demonstrated that Ad transduction of glioblastoma and carcinoma cells can be increased by complexing the virus with a recombinant tandem a-fiber/a-EGFR scFv (Haisma et al. 2000) . Our group reported in 2001 that a scDb with specificities for the Ad fiber and Endoglin, which is expressed on proliferative endothelium, facilitated targeted transduction of endothelial cells (Nettelbeck et al. 2001) . In contrast to the tandem scFv, which was expressed in eukaryotic cells, the scDb was produced in bacteria. Ad transduction was also targeted to gastric cancer cells with a tandem scFv adapter binding to EpCAM (Heideman et al. 2002) , to DCs with a CD40-binding tandem scFv adapter (Brandao et al. 2003) , to melanoma cells using a scDb adapter binding the melanoma surface antigen HMWMAA ), or to breast cancer cells with either a tandem scFv or a scDb binding to CEA (Korn et al. 2004 ). For improved de-targeting, "receptor-blind" Ad mutants were combined with tandem scFv or scDb adapters that were derived from a-fiber scFvs that retained binding to mutant fibers Nettelbeck et al. 2004; Carette et al. 2007 ). These Ad vectors could not bind CAR, even when individual fiber molecules were not protected after complexation with adapters. In consequence, this strategy of combined genetic/immunological tropism-modification implements a further increase in selectivity of gene transfer. Recombinant antibody-derived adapters for targeting adenoviral transduction were also obtained by fusion of a-fiber scFv to ligand proteins (EGF or uPAR, Watkins et al. 1997; Harvey et al. 2010) or to ligand peptides (Nicklin et al. 2000) . Alternatively, cell-binding scFvs (a-c-erbB2, a-CD40 or a-FcgRI) were fused to monomeric or trimeric soluble CAR Pereboev et al. 2002; Kim et al. 2002; Sapinoro et al. 2007 ). Such sCAR-derived adapters offer the advantage of improving affinity to fiber by sCAR trimerization; however, they naturally cannot bind to "receptorblind" fiber-mutant viruses. These strategies also demonstrated that the adapter, besides targeting gene transfer, might also influence the outcome of gene therapy in different ways: adenoviral gene transfer to DCs by CD40-binding adapters, but not by the FcgRI-binding adapter resulted in DC activation, thus influencing the type of immune response (immunization versus tolerization, Tillman et al. 1999; Sapinoro et al. 2007 ).

In vitro studies with adapter molecules, including various bispecific antibodies, have clearly proven that viral cell entry can be re-directed via novel cell surface receptors, thus reprogramming virus tropism. This has been demonstrated in established cell cultures, freshly purified normal and tumor cells and in tissue explants, as for the demonstration of DC-targeted gene transfer in skin explants (de Gruijl et al. 2002) . What are possible applications of bispecific antibodies and other antibody-derived gene transfer adapters? First, due to their modular composition and the opportunity to rapidly (in comparison with genetically engineered viruses) produce new adapters by chemical or genetic means, they facilitate the analysis, comparison, and screening of cell surface molecules for their feasibility as targets for viral gene transfer. Second, applications of adapters for ex vivo gene therapy are of interest. An example is genetic vaccination of cancer or infectious diseases by ex vivo gene transfer into DCs isolated from patients. Gene therapy of inherited diseases by ex vivo gene transfer into hematopoietic (stem) cells is a further application. Here, however, retroviral vectors are preferred over Ad vectors, as they facilitate stable gene transfer and thus prolonged gene correction or replacement (Table 18 .2). Of note, adaptertargeting of retroviral gene transfer has been demonstrated recently (see below). Most gene therapy applications, however, require in vivo gene transfer. For establishing adapters for targeting gene transfer, in vivo extensive studies on the stability, efficiency, and selectivity of adapter-vector complexes after in vivo application are needed. Whereas rigorous studies for the evaluation of pharmacologic and therapeutic parameters of adapter-targeted gene transfer are still to be done, initial studies have shown efficacy of adapter-targeting in vivo. In an effort to facilitate gene therapy of pulmonary vascular disease, Reynolds and colleagues investigated a Fab-mAb conjugate adapter that binds angiotensinconverting enzyme (ACE) for targeting of Ad gene transfer to the lung endothelium in rats (Reynolds et al. , 2001 . By systemic application of adapter-bound or uncomplexed Ad vector, it was shown that this adapter increased gene transfer to the lung by more than 20-fold. Importantly, gene transfer was directed to endothelial cells. Moreover, gene transfer to the liver, the organ responsible for most Adinduced side effects, was reduced more than 80%. Hence, this study demonstrated both systemic stability of the adapter-vector complex and adapter-dependent vector de-and re-targeting in vivo. For the Fab-FGF2 adapter, several studies in mice showed adapter-dependent reduction of liver transgene expression after systemic injection of Ad vectors and reduced toxicity of Ad-mediated genetic prodrug activation therapy. Furthermore, this adapter increased therapeutic activity of Ad-mediated genetic prodrug activation of peritoneal malignancies, when the Ad vectors were injected intraperitoneally (Rancourt et al. 1998; Gu et al. 1999; Printz et al. 2000) . In vivo stability of adaptor-vector complexes has also been demonstrated for recombinant proteins. Trimeric, but not monomeric sCAR significantly blocked liver gene transfer by Ads after systemic application of the sCAR-Ad vector complex into mice (Kim et al. 2002) . However, in a different study, a sCAR-scFv adapter targeting CEA also reduced liver transduction by Ad vectors after systemic injection of adapter-virus complexes into mice (Li et al. 2007) . After systemic injection, this adapter also increased adenoviral transduction of CEApositive, but reduced transduction of CEA-negative tumors that were grafted to mouse livers. Furthermore, a trimeric derivative of the sCAR-CEA adapter mediated improved targeting of adenoviral gene transfer in vitro and in vivo. In combination with transcriptional targeting using the cox-2 promoter, this trimeric adapter increased therapeutic activity and at the same time reduced liver toxicity of genetic prodrug activation therapy with HSV-tk/GCV (Li et al. 2009 ). Studies with sCAR-EGF and trimeric sCAR-mCD40L confirm the re-targeting properties of sCAR-derived adapters in vivo (Liang et al. 2004; Huang et al. 2007 ). In addition to facilitating selective gene transfer, targeting adenoviral cell binding and entry is of interest also for improving oncolytic Ads. Toward this end, adapter molecules are of interest to re-direct the injected virus to target tumors. To also allow for targeting of progeny viruses of oncolytic Ads produced in patients' tumors, genes encoding recombinant bispecific adapters have been inserted into the genome of these viruses. Using a tandem scFv with specificity for the Ad fiber and EGFR, van Beusechem and co-workers demonstrated increased viral spread and oncolysis in two-and three-dimensional tumor cell cultures (van Beusechem et al. 2003; Carette et al. 2007 ).

Although most widely investigated for Ad vectors, adapters have been also shown to facilitate targeted cell entry of other viruses. Adeno-associated viruses (AAV) are small non-enveloped viruses that are frequently used for diverse gene therapy applications (Buning et al. 2008) . Tropism-modification of AAV vectors was achieved with a bispecific Fab/Fab antibody conjugate. The adapter with specificity for the virus capsid and for integrins facilitated gene transfer into megakaryocytes, which are not permissive to unmodified AAV vectors (Bartlett et al. 1999) . For non-human coronaviruses, enveloped RNA viruses that naturally do not enter human cells, infectivity for human cancer cells was established with a bispecific tandem scFv with specificities for a coronavirus surface glycoprotein and EGFR (Wurdinger et al. 2005a, b) . The idea of this approach was to selectively kill tumor cells by lytic virus infection rather than viral gene transfer. Similar results were obtained using a recombinant adapter built of soluble coronavirus receptor fused to the EGFR-binding scFv (Wurdinger et al. 2005a, b) . Newcastle disease virus, which is in development for viral oncolysis and gene therapy, has been re-targeted using a recombinant adapter built of a virus-binding scFv and IL-2 (Bian et al. 2005 (Bian et al. , 2006 . Retroviruses are enveloped RNA viruses, which insert their genome after reverse transcription into the chromosome of infected cells. Therefore, retroviral vectors facilitate long-term gene transfer which is especially suitable for gene correction therapy of monogenetic diseases.

Adapter targeting of retrovirus cell entry was reported for recombinant proteins built of the virus receptor extracellular domain fused to EGF, VEGF, or an EGFRspecific scFv (Snitkovsky and Young 1998; Boerger et al. 1999; Snitkovsky et al. 2000 Snitkovsky et al. , 2001 .

18.3 Gene Transfer as a Tool for Antibody Therapy: Genetic Antibody Delivery

Gene therapy can be exploited for expressing antibodies in patients, which might be advantageous for achieving sustained and/or efficient antibody concentrations and/ or a favorable antibody biodistribution by local expression. Thus, gene therapy is a tool of interest to overcome rapid antibody clearance or poor access to tumors as reported for antibodies that are injected as proteins. Genetic antibody therapy can be implemented by in vivo or ex vivo gene transfer (Fig. 18.3) , i.e., by direct injection of the gene transfer vector into patients or by gene transfer in cultures of previously isolated cells followed by injection of the resulting genetically engineered cells, respectively. Dependent on the design of the gene transfer vector, genetic antibody application can be transient or permanent, constitutive or inducible, targeted or ubiquitous. For example, retroviral vectors allow for stable gene transfer, inducible promoters facilitate control of antibody expression, and targeted vectors can direct gene transfer to specific cell types (see Sects. 18.1.2 and 18.1.3). Therefore, gene therapy possesses high potential and flexibility for implementing improved antibody delivery for specific applications. However, this area of research is still in its infancy and more widespread investigations are warranted.

With the advent of recombinant DNA technology it became possible to establish novel strategies for antibody production and to engineer antibody properties (for example affinity maturation and humanization), formats (single chain fragments), and fusion proteins (immunotoxins). Recombinant antibodies have been frequently produced in bacteria, but gene transfer into eukaryotic cells has also been utilized for in vitro production of immunoglobulins, antibody fragments or antibody fusion proteins. Having established the engineering of recombinant gene constructs for eukaryotic antibody expression, also the in vivo production of antibodies became feasible. Examples are the expression of functional recombinant MAbs in mice after transfer of genetically engineered cells (Noel et al. 1997) or after in vivo gene transfer with an adenoviral or AAV vector (Noel et al. 2002; Jiang et al. 2006; Watanabe et al. 2009; Lewis et al. 2002; Fang et al. 2005 Fang et al. , 2007 Skaricic et al. 2008; De et al. 2008; Ho et al. 2009 ). Toward this end, Fang and coworkers optimized antibody production: they expressed the heavy and light chains of the MAb at equal amounts from a single open reading frame using a "ribosomal skip" sequence. Thereby, serum levels of >1 mg/ml antibody for extended time periods were obtained in mice after injection of a single dose of AAV vector. In a subsequent study, the same group demonstrated that by using an inducible promoter, serum antibody levels after in vivo gene transfer can be repeatedly shut off and on (Fang et al. 2007 ). This represents a promising strategy to increase safety and/or facilitate dose adaptation in potential future clinical applications of genetic Fig. 18. 3 Gene therapy as a tool for antibody delivery: Genetic antibody therapy. For genetic antibody delivery antibody genes, which can be engineered to match specific purposes, are incorporated into gene transfer vectors. These vectors are either directly injected into patients (in vivo gene therapy) or are used for gene transfer into cells previously purified from a patient followed by re-injection of the engineered cells into the patient (ex vivo gene therapy). The antibodies are produced in the patient from cells genetically modified by in vivo or ex vivo gene transfer. Dependent on the vector design, antibody production can be transient or prolonged, constitutive or inducible and show local or systemic activity antibody delivery. De and co-workers combined genetic delivery of a MAb gene by AAV and Ad vectors to achieve both rapid (Ad) and persistent (AAV) antibody production . Functional expression in vivo was also demonstrated for recombinant antibody fragments or fusion proteins that contain such fragments after adenoviral gene transfer (Whittington et al. 1998; Arafat et al. 2002; Afanasieva et al. 2003; Kasuya et al. 2005; Liu et al. 2010) . The expression of chimeric antigen receptors by T cells and subsequent adoptive T cell therapy is another important application of genetic antibody delivery.

Á lvarez-Vallina and team have developed genetic delivery of bispecific antibodies by engineered cells. In 2003, they reported anti-tumor activity for a bispecific diabody expressed in vivo from irradiated, genetically engineered 293T cells (Blanco et al. 2003) . They produced stably transfected 293T cells secreting a diabody with specificity for both CEA and CD3. A second cell line additionally secreted a bivalent CEA-specific diabody fused to the extracellular domain of B7-1. After co-injection with CEA-positive tumor cells into mice, these genetically engineered cells showed anti-tumor activity compared with co-injection of control 293T cells. Subsequent to this proof-of-principle study, the same group engineered a lentiviral gene transfer vector encoding the CEA-CD3 diabody (Compte et al. 2007 ). This vector facilitated the transduction of different types of hematopoietic cells that showed prolonged secretion of active diabody in vitro and antitumor activity in vivo. In a follow-up study, the group demonstrated that also the implantation of lentivirally transduced endothelial cells into mice resulted in prolonged production of the CEA/CD3 diabody with therapeutic activity (Compte et al. 2010 ). This study aims at a therapeutic regimen that allows for the production of therapeutic antibodies from neovessels that have incorporated ex vivo engineered endothelial cells.

Genetic delivery of bispecific antibodies has also been reported for intracellular applications: cell surface localization of two membrane proteins, VEGFR2 and Tie-2, could be blocked by expression of a corresponding bispecific, tetravalent antibody targeted to the endoplasmic reticulum (Jendreyko et al. 2003) . This intracellular bispecific antibody showed anti-angiogenic activity in vitro, which was superior to monovalent control antibodies. A similar construct with specificity for VEGFR2 and Tie-2 mediated anti-angiogenic and anti-tumor activity in vivo after adenoviral gene transfer (Jendreyko et al. 2005) .

Proof of principle has been demonstrated in several cell culture studies and animal models for both the utility of bispecific antibodies for targeting gene therapies and the feasibility of gene transfer for delivering recombinant bispecific antibodies.

Based on this fundamental work, bispecific antibody adapters and gene transfer technologies should now be considered for improving therapeutic regimens in gene therapy and antibody therapy, respectively. Cooperation between antibody engineers and gene therapists are warranted to further develop bispecific antibodies and gene transfer vectors for this purpose.

Single-chain antibody and its derivatives directed against vascular endothelial growth factor: application for antiangiogenic gene therapy

Effective single chain antibody (scFv) concentrations in vivo via adenoviral vector mediated expression of secretory scFv

Dual targeting of adenoviral vectors at the levels of transduction and transcription enhances the specificity of gene expression in cancer cells

Targeted adeno-associated virus vector transduction of nonpermissive cells mediated by a bispecific F(ab'gamma)2 antibody

Retrovirus vectors: toward the plentivirus?

Selective gene transfer in vitro to tumor cells via recombinant Newcastle disease virus

In vivo efficacy of systemic tumor targeting of a viral RNA vector with oncolytic properties using a bispecific adapter protein

Retargeting to EGFR enhances adenovirus infection efficiency of squamous cell carcinoma

Induction of human T lymphocyte cytotoxicity and inhibition of tumor growth by tumor-specific diabody-based molecules secreted from gene-modified bystander cells

Retroviral vectors preloaded with a viral receptor-ligand bridge protein are targeted to specific cell types

CD40-targeted adenoviral gene transfer to dendritic cells through the use of a novel bispecific single-chain Fv antibody enhances cytotoxic T cell activation

Targeted gene-delivery strategies for angiostatic cancer treatment

Exploiting and antagonizing microRNA regulation for therapeutic and experimental applications

Recent developments in adeno-associated virus vector technology

A conditionally replicating adenovirus with strict selectivity in killing cells expressing epidermal growth factor receptor

Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy

Armed replicating adenoviruses for cancer virotherapy

Inhibition of tumor growth in vivo by in situ secretion of bispecific anti-CEA x anti-CD3 diabodies from lentivirally transduced human lymphocytes

Factory neovessels: engineered human blood vessels secreting therapeutic proteins as a new drug delivery system

Prolonged maturation and enhanced transduction of dendritic cells migrated from human skin explants after in situ delivery of CD40-targeted adenoviral vectors

Rapid/sustained anti-anthrax passive immunity mediated by co-administration of Ad/AAV

Targeting cancer by transcriptional control in cancer gene therapy and viral oncolysis

Fifteen years of gene therapy based on chimeric antigen receptors: "are we nearly there yet?

Targeted gene delivery by tropism-modified adenoviral vectors

Stable antibody expression at therapeutic levels using the 2A peptide

An antibody delivery system for regulated expression of therapeutic levels of monoclonal antibodies in vivo

Targeted gene delivery to Kaposi's sarcoma cells via the fibroblast growth factor receptor

Regulatable gene expression systems for gene therapy applications: progress and future challenges

TRAIL gene therapy: from preclinical development to clinical application

Fibroblast growth factor 2 retargeted adenovirus has redirected cellular tropism: evidence for reduced toxicity and enhanced antitumor activity in mice

Tumor-specific gene transfer via an adenoviral vector targeted to the pan-carcinoma antigen EpCAM

Targeting of adenoviral vectors through a bispecific single-chain antibody

Retargeted adenoviral cancer gene therapy for tumour cells overexpressing epidermal growth factor receptor or urokinase-type plasminogen activator receptor

An adenovirus vector with a chimeric fiber incorporating stabilized single chain antibody achieves targeted gene delivery

Selective gene delivery toward gastric and esophageal adenocarcinoma cells via EpCAM-targeted adenoviral vectors

Selective gene transfer into primary human gastric tumors using epithelial cell adhesion molecule-targeted adenoviral vectors with ablated native tropism

Growth inhibition of an established A431 xenograft tumor by a full-length anti-EGFR antibody following gene delivery by AAV

Enhancement of adenovirus vector entry into CD70-positive B-cell Lines by using a bispecific CD70-adenovirus fiber antibody

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

Phenotypic knockout of VEGF-R2 and Tie-2 with an intradiabody reduces tumor growth and angiogenesis in vivo

Gene therapy using adenovirus-mediated full-length anti-HER-2 antibody for HER-2 overexpression cancers

New aspects in vascular gene therapy

Adenovirus targeting to c-erbB-2 oncoprotein by single-chain antibody fused to trimeric form of adenovirus receptor ectodomain

Passive immunotherapy for anthrax toxin mediated by an adenovirus expressing an anti-protective antigen single-chain antibody

Selectivity of TAG-72-targeted adenovirus gene transfer to primary ovarian carcinoma cells versus autologous mesothelial cells in vitro

Targeting adenoviral vectors by using the extracellular domain of the coxsackie-adenovirus receptor: improved potency via trimerization

Update on gene therapy for immunodeficiencies

Recombinant bispecific antibodies for the targeting of adenoviruses to CEA-expressing tumour cells: a comparative analysis of bacterially expressed single-chain diabody and tandem scFv

Generation of neutralizing activity against human immunodeficiency virus type 1 in serum by antibody gene transfer

Adenovirus tumor targeting and hepatic untargeting by a coxsackie/adenovirus receptor ectodomain anticarcinoembryonic antigen bispecific adapter

Combined transductional untargeting/retargeting and transcriptional restriction enhances adenovirus gene targeting and therapy for hepatic colorectal cancer tumors

Noninvasive of adenovirus tumor retargeting in living subjects by a soluble adenovirus receptor-epidermal growth factor (sCAR-EGF) fusion protein

Advances in viral-vector systemic cytokine gene therapy against cancer

Gene therapy in haemophilia -going for cure?

Biology of adenovirus and its use as a vector for gene therapy

Differential susceptibility of primary and established human glioma cells to adenovirus infection: targeting via the epidermal growth factor receptor achieves fiber receptor-independent gene transfer

Targeting of adenovirus to endothelial cells by a bispecific singlechain diabody directed against the adenovirus fiber knob domain and human endoglin (CD105)

Retargeting of adenoviral infection to melanoma: combining genetic ablation of native tropism with a recombinant bispecific single-chain diabody (scDb) adapter that binds to fiber knob and HMWMAA

Selective targeting of gene transfer to vascular endothelial cells by use of peptides isolated by phage display

In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells

High in vivo production of a model monoclonal antibody on adenoviral gene transfer

Recent progress in the battle between oncolytic viruses and tumours

Coxsackievirus-adenovirus receptor genetically fused to anti-human CD40 scFv enhances adenoviral transduction of dendritic cells

Suicide genes for cancer therapy

Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX

Fibroblast growth factor 2-retargeted adenoviral vectors exhibit a modified biolocalization pattern and display reduced toxicity relative to native adenoviral vectors

Basic fibroblast growth factor enhancement of adenovirusmediated delivery of the herpes simplex virus thymidine kinase gene results in augmented therapeutic benefit in a murine model of ovarian cancer

A targetable, injectable adenoviral vector for selective gene delivery to pulmonary endothelium in vivo

Combined transductional and transcriptional targeting improves the specificity of transgene expression in vivo

DNA vaccines: precision tools for activating effective immunity against cancer

Use of a novel cross-linking method to modify adenovirus tropism

Ocular gene therapy: an evaluation of recombinant adenoassociated virus-mediated gene therapy interventions for the treatment of ocular disease

Enhanced transduction of dendritic cells by FcgammaRI-targeted adenovirus vectors

Retargeting of adenoviral vectors to neurons using the Hc fragment of tetanus toxin

Applications of lentiviral vectors for shRNA delivery and transgenesis

Genetic delivery of an anti-RSV antibody to protect against pulmonary infection with RSV

Dendritic cell-based cancer gene therapy

Cell-specific viral targeting mediated by a soluble retroviral receptor-ligand fusion protein

A TVA-single-chain antibody fusion protein mediates specific targeting of a subgroup A avian leukosis virus vector to cells expressing a tumor-specific form of epidermal growth factor receptor

Targeting avian leukosis virus subgroup A vectors by using a TVA-VEGF bridge protein

Maturation of dendritic cells accompanies high-efficiency gene transfer by a CD40-targeted adenoviral vector

Adenoviral vectors targeted to CD40 enhance the efficacy of dendritic cell-based vaccination against human papillomavirus 16-induced tumor cells in a murine model

Molecular adaptors for vasculartargeted adenoviral gene delivery

Efficient and selective gene transfer into primary human brain tumors by using single-chain antibody-targeted adenoviral vectors with native tropism abolished

Conditionally replicative adenovirus expressing a targeting adapter molecule exhibits enhanced oncolytic potency on CAR-deficient tumors

Efficient incorporation of a functional hyper-stable single-chain antibody fragment protein-IX fusion in the adenovirus capsid

Genetic delivery of bevacizumab to suppress vascular endothelial growth factor-induced high-permeability pulmonary edema

AAVrh.10-mediated genetic delivery of bevacizumab to the pleura to provide local anti-VEGF to suppress growth of metastatic lung tumors

The 'adenobody' approach to viral targeting: specific and enhanced adenoviral gene delivery

Gene therapy progress and prospects: electroporation and other physical methods

Recombinant adenoviral delivery for in vivo expression of scFv antibody fusion proteins

Targeted adenovirus gene transfer to endothelial and smooth muscle cells by using bispecific antibodies

Breaking the bonds: non-viral vectors become chemically dynamic

Soluble receptor-mediated targeting of mouse hepatitis coronavirus to the human epidermal growth factor receptor

Targeting non-human coronaviruses to human cancer cells using a bispecific single-chain antibody