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A Chimeric Fusion Protein Containing Transforming Growth Factor-Α

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A Chimeric Fusion Protein Containing Transforming Growth Factor-Α Gene Therapy (1998) 5, 521–530 1998 Stockton Press All rights reserved 0969-7128/98 $12.00 http://www.stockton-press.co.uk/gt A chimeric fusion protein containing transforming growth factor-␣ mediates gene transfer via binding to the EGF receptor J Fominaya1,2, C Uherek1 and W Wels1 1Institute for Experimental Cancer Research, Tumor Biology Center, Breisacher Strasse 117, D-79106 Freiburg, Germany Fusion proteins engineered to incorporate distinct functions binding to EGF receptors. Complexes of the chimeric pro- which co-operate in mediating the cell-type specific uptake tein and plasmid DNA carrying a luciferase reporter gene, and intracellular delivery of DNA present an attractive after condensation with poly-L-lysine resulted in an up to approach for the development of self-assembling vector 150-fold increase in reporter gene expression in EGF systems for targeted gene transfer. Here we have chosen receptor expressing cells in comparison to poly-L-lysine– the EGF receptor overexpressed in many human tumors DNA complexes alone. While in COS-1 cells no of epithelial origin as a target for a novel modular fusion additional endosome escape activity was required, in protein. We have fused a cDNA fragment of the human A431 cells gene delivery was dependent on the simul- EGF receptor ligand TGF-␣ to sequences encoding the taneous presence of the endosome destabilizing translocation domain of Pseudomonas exotoxin A as an reagent chloroquine indicating that cell-type specific fac- endosome escape activity, and the DNA-binding domain of tors such as different intracellular routing of protein–DNA the yeast GAL4 transcription factor. Upon bacterial complexes greatly influence transfection efficiency. expression, this TEG fusion protein displayed specific Keywords: gene therapy; non-viral gene delivery; fusion protein; TGF-␣; EGF receptor; Pseudomonas exotoxin A; GAL4 Introduction distinct activity required for DNA-binding, cell recog- nition and intracellular delivery. Such fusion proteins in The development of vectors for target-cell specific gene contrast to chemical conjugates can be produced in suit- delivery is a major goal of gene therapeutic strategies. able expression systems in their final form and the Thereby nonviral gene delivery vectors are gaining resulting products are generally homogeneous in their 1,2 increasing interest. While they are still relatively inef- composition. We have recently described such a chimeric ficient, they might present a suitable alternative to viral multidomain fusion protein which facilitates target-cell vectors since they overcome major disadvantages of specific gene delivery and is based on the molecular viruses such as the lack of cell type specificity with organization of the bacterial Pseudomonas exotoxin A regard to their infectivity, the restriction in the size of (ETA).6 incorporated DNA, and safety considerations due to the The 66 kDa exotoxin A consists of three functional presence of viral genomic sequences. Progress has been domains defined by crystal structure and deletion analy- made in the understanding of how individual activities sis.8,9 Upon cell binding via the N-terminal cell recog- of viruses can be mimicked and methodologies have been nition domain (Ia) and receptor-mediated internalization, developed which allow the combination of different func- ETA is cleaved by a cellular protease within the internal tions required for gene transfer into an artificial complex. domain II (translocation domain) resulting in an N-ter- Molecular conjugates which employ antibodies or minal 28 kDa and a C-terminal 37 kDa fragment. After other ligands chemically coupled to polycations to reduction of a disulfide bond, the C-terminal fragment deliver DNA into cells via receptor-mediated endocytosis containing the enzymatic domain III translocates to the constitute an important group of such target-cell specific, cytoplasm where it ADP-ribosylates and inactivates 3,4 nonviral vectors. Another attractive approach for the eukaryotic elongation factor 2.10 In the 5EG fusion pro- design of modular self-assembling systems for gene tein,6 the natural cell binding domain of ETA was delivery is based on fusion proteins engineered to incor- replaced by a tumor-specific single chain antibody porate several co-operating functions in a single polypep- domain. Likewise, the C-terminal enzymatic domain of 5–7 tide chain. Each of these domains can account for a the toxin was exchanged with the DNA-binding domain of the yeast GAL4 transcription factor for the interaction with plasmid DNA. The internal translocation domain of Correspondence: W Wels ETA was retained as an endosome escape function. The 2Present address: Department of Immunology and Oncology, Centro ´ ´ Nacional de Biotecnologıa/CSIC, Universidad Autonoma de Madrid, resulting chimeric molecule was non-toxic, exhibited an Campus Cantoblanco, Madrid E-28049, Spain altered target–cell tropism, and functioned as a cell- Received 5 September 1997; accepted 28 November 1997 specific DNA carrier. Cell-specific gene transfer mediated by a TGF-␣ fusion protein J Fominaya et al 522 Due to this modular organization, similar carrier pro- teins with modified activities, eg different target cell specificity, might be obtained by replacing individual protein domains. This could assist in the development of gene transfer strategies optimized for distinct target cell populations. In order to explore the flexibility of this con- cept, we have constructed and characterized a multi- domain fusion protein which employs the natural EGF receptor ligand transforming growth factor (TGF)-␣ as a cell targeting domain. EGF receptor overexpression has been observed in a high percentage of human carcinomas including glioblastoma, cancer of the lung, breast, head and neck and bladder, and correlates with an unfavorable prognosis for the patients.11 Therefore the EGF receptor constitutes an attractive target for directed tumor therapy which includes gene therapeutic strategies.12–14 We have fused a human TGF-␣ cDNA fragment to sequences encoding the ETA translocation domain and the GAL4 DNA-binding domain. Upon bacterial expression and purification, the resulting chimeric pro- tein termed TEG displayed specific binding to EGF recep- tor expressing cells. Complexes of the fusion protein with plasmid DNA carrying a luciferase reporter gene and a GAL4 specific recognition sequence, after condensation with poly-l-lysine were able to transfect EGF receptor expressing cells in vitro. Thereby transfection efficiency and dependence on the endosomolytic reagent chloro- Figure 1 (a) Schematic representation of the TEG fusion gene in the E. quine varied in different cell lines demonstrating the coli expression plasmid pWF47-TEG. The inducible tac promoter controls importance of cell type-specific characteristics for suc- the expression of the chimeric TEG protein which consists of an N-ter- cessful gene delivery. minal E. coli ompA signal peptide (SP), the synthetic FLAG epitope (F), amino acids 1 to 50 of human TGF-␣, a polyhistidine tag (H), amino acids 252 to 366 of Pseudomonas exotoxin A (translocation domain) Results amino acids 2 to 147 of the yeast GAL4 protein (DNA-binding domain) and a C-terminal KDEL endoplasmic reticulum retention signal. (b) SDS- Construction and bacterial expression of the TEG DNA PAGE analysis of the TEG protein purified from bacterial lysate. Lane 1, carrier protein bacterial lysate cleared by ultracentrifugation; lane 2, flow through from 2+ DNA fragments encoding amino acids 1 to 50 of human aNi -saturated chelating Sepharose column; lane 3, wash with 50 mm TGF-␣, domain II (translocation domain) of Pseudomonas imidazole; lane 4, eluate with 250 mm imidazole; lane 5, immunoblot analysis of purified TEG protein with a MAb specific for the GAL4 DNA exotoxin A, and the DNA binding domain of the yeast binding domain; M, molecular weight standards. transcriptional activator GAL4 were derived from pre- viously described plasmids by restriction enzyme diges- tion and assembled into a single open reading frame in Binding properties of the TEG fusion protein the bacterial expression vector pFLAG-1. The resulting To investigate the functionality of the TGF-␣ domain, the plasmid pWF47-TEG encodes under the control of the binding of purified TEG protein to the recombinant EGF IPTG inducible tac promoter the chimeric fusion protein receptor was analyzed by ELISA. Purified TEG at concen- termed TEG which is schematically shown in Figure 1a. trations ranging from 0.05 to 333 nm was added to the It consists of an N-terminal E. coli ompA signal peptide, wells of 96-well plates coated with recombinant extra- a synthetic FLAG epitope, human TGF-␣, a cluster of six cellular domain of the EGF receptor, the plates were incu- histidine residues facilitating the purification of the bated at room temperature for 1 h and specifically bound recombinant protein via Ni2+ affinity chromatography, protein was determined. The results are shown in Figure amino acids 252 to 366 of Pseudomonas exotoxin A, and 2a. TEG bound to the extracellular portion of the EGF amino acids 2 to 147 of the yeast GAL4 protein. At the receptor in a saturable fashion. This interaction with the C-terminus, the fusion protein carries a KDEL signal EGF receptor was dependent on the TGF-␣ domain of which is functionally equivalent to the C-terminal TEG since the presence of a nine- to 40-fold molar excess sequence of exotoxin A required for intracellular routing of TGF␣-ETA, a recombinant toxin containing TGF-␣,16 and full activity of
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