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Recombinant Single-Chain Antibodies with Various Oligopeptide Tails for Targeted Gene Delivery

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Recombinant Single-Chain Antibodies with Various Oligopeptide Tails for Targeted Gene Delivery Gene Therapy (2003) 10, 781–788 & 2003 Nature Publishing Group All rights reserved 0969-7128/03 $25.00 www.nature.com/gt RESEARCH ARTICLE Recombinant single-chain antibodies with various oligopeptide tails for targeted gene delivery M Suzuki, A Takayanagi and N Shimizu Department of Molecular Biology, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan The single-chain antibody (scFv) made by recombinant DNA charged tails were efficiently expressed and secreted into the technology is one of the most useful tools for basic research culture medium. Addition of high salt into the yeast culture and clinical applications. To develop a novel targeted gene increased their secretion. Purification procedure was estab- delivery method, we engineered the scFv gene for the lished for the scFv with the longest negatively charged tail antibody against human epidermal growth factor (EGF) (D4S Â 5), yielding 5 mg/l with a purity of over 95%. The receptor by connecting with DNA sequences for various scFv-D4S Â 5 was designated as a recombinant immuno- oligopeptides with negative or positive charges. The resulting porter, which was then mixed with plasmid DNA and recombinant genes encoding artificial scFv with negative or polyethylenimine (PEI). The resulting DNA/PEI/immunopor- positive tails were expressed in Escherichia coli and yeast ter complex (designated recombinant immunogene) exhib- Pichia pastris. In E. coli, all the scFv with negatively charged ited efficient gene delivery to EGF receptor overexpressing tails were expressed but mainly as an insoluble form, A431 tumor cells. whereas those with positively charged tails were barely Gene Therapy (2003) 10, 781–788. doi:10.1038/sj.gt.3301952 expressed. In yeast P. pastris, all the scFv with negatively Keywords: EGF receptor; recombinant immunoporter; scFv antibody; Pichia pastris; targeting gene therapy; immunogene Introduction form Fab-immunoporter.10,11 Affinity complex was formed by mixing the Fab immunoporter and a The viral vectors constructed for gene delivery provide therapeutic DNA. The resulting Fab immunoporter highly efficient transfection and thus they have been carrying the herpes TK gene was effective in preventing often employed for gene therapy to cure various the growth of EGF receptor-overexpressing tumor cells diseases. However, only a limited number of patients both in vitro and in vivo.12 have received true benefits from the treatment with the It has been well established that a minimal structural current gene therapy. The problems associated with viral component required for antigen-binding activity of vectors include the immune response upon repeated the antibody resides in the Fv fragment.13 Consequently, administration and difficulties to maintain the specificity the recombinant gene was constructed to produce of targeting certain types of cells and tissues. Thus, the artificial single-chain antibody (scFv) by bridging current efforts have been placed to further improve viral VL and VH domains with a linker peptide.14 vectors by conjugating with unique promoter and/or The recombinant scFv gene is easy to manipulate enhancer that are active in the limited types of cells and for further modification and its expression has been tissues.1,2 Alternatively, certain biological activities such successful in Escherichia coli, yeast and mammalian as ligands, receptors, antigens and antibodies have been cells.15–17 Along this line, we have produced a recombi- challenged to select cell types.3–7 These novel approaches nant scFv reactive to EGF receptor and confirmed that it have made substantial improvement, but yet further is competitive with monoclonal antibody B4G7 for the innovation has been in demand. binding to cell surface EGF receptor (Takayanagi A, To aim at the targeted delivery of a therapeutic gene manuscript in preparation). This encouraged us to apply without using viral vectors, we previously developed a recombinant scFv technology for the immunogene novel immunogene system using the immunoporter, system. The major strategic point on the development which is a monoclonal antibody against EGF receptor of recombinant scFv immunoporter is to improve conjugated with cationic polylysine chain.8,9 In the significantly the gene transfer efficiency while maintain- improved immunogene system, the Fab fragment of the ing the ability to recognize a specific antigen. We thus monoclonal antibody was conjugated with polylysine to attempted to produce several recombinant antibodies with various oligopeptide tails of negative or positive charges. In this study, we provide evidence that the recombi- Correspondence: Dr N Shimizu, Department of Molecular Biology, Keio nant immunogene serves as a highly efficient gene University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan delivery system and will be useful for the future gene Received 20 May 2002; accepted 5 November 2002 therapy of various diseases. Recombinant immunogene system utilizing scFv M Suzuki et al 782 Results of these recombinant vectors was introduced into two different E. coli strains AD494(DE3) and AD494(DE3)- Expression of recombinant antibody with charged tails pLysS, and four independent clones bearing recombinant in E. coli antibody genes were isolated. These transformed E. coli We constructed various recombinant genes that encode clones were independently cultured and their expression scFv with various oligopeptide tails of positive or was induced with IPTG. Cells were collected and negative charges (Figure 1). Positively charged tails disrupted, then soluble periplasmic fractions and in- include K8S, R8S, R4S Â 2 and R4S Â 5 whereas nega- soluble fractions were processed for Western blot tively charged tails include D8S, E8S, D4S Â 2 and analysis (Figure 2). All the recombinant antibodies with D4S Â 5. Each of these recombinant genes was cloned charged tails were expressed in the strain AD494(DE3) as into a high copy vector (pET22bhc) designed to produce their expected sizes, but much of them were found in the secretory proteins with pelB signal sequence. Then, each insoluble fractions (Figure 2a, b). Exceptionally, the recombinant antibody with the longest negatively charged tail (D4S Â 5) was secreted into the medium. Expression of the recombinant antibodies in the other strain AD494(DE3)pLysS was much less than that in the AD494(DE3) (Figure 2c, d). Expression of recombinant antibody with charged tails in yeast Pichia pastris Next, we attempted to express recombinant antibodies with charged tails in the yeast Pichia pastris. The sequences of various recombinant genes encoding scFv with various charged tails were introduced into the P. pastris genome by electroporation. Three transformed clones that bear the recombinant antibody genes were S Figure 1 Construction of the gene for scFv with various oligopeptide tails. isolated from both Mut (methanol utilization slow) and + ScFv consists of VH-linker-VL domains and two units of His-tag (six Mut (methanol utilization plus) strains of P. pastris. consecutive His residues) in a vector. LK indicates four amino-acid Expression of recombinant antibody was induced by residues (GGGS). Positively charged tails include K8S, R8S, R4S Â 2 and resuspending cells in a medium containing 0.5% metha- R4S Â 5, whereas negatively charged tails include D8S, E8S, D4S Â 2 and nol. The recombinant antibodies with negatively charged D4S Â 5. K8S and R8S consist of eight consecutive lysine or arginine S residues connected to one serine residue. R4S Â 2 indicates that R4S unit tails were expressed in all the Mut clones, whereas consisting of four arginine residues and one serine residue is repeated the recombinant antibody with positively charged tails S + twice. Amino acids in the tail domains are indicated as single letter; K: were barely expressed in both Mut and Mut clones lysine, R: arginine, S: serine, D: asparagine and E: glutamine. (Figure 3). Figure 2 Expression of scFv with various charged tails in E. coli. Four E. coli clones bearing recombinant scFv constructs were cultured and their expression was induced with 1 mM IPTG at 371C for 3 h. Cell lysates were prepared from: (1) whole cells before induction, (2) whole cells after induction, (3) medium, (4) periplasms and (5) insoluble fractions. (a) and (b) : AD494(DE3), (c) and (d) : AD494(DE3)pLysS. Extracts were analyzed on a 15% SDS- PAGE and transferred to a PVDF membrane. The immunostaining was carried out using anti-5 Â His antibody as first antibody and alkaline phosphatase- conjugated anti-mouse IgG antibody as second antibody. (À): E. coli clones bearing scFv gene without charged tail; scFv(À): scFv without charged tail. Gene Therapy Recombinant immunogene system utilizing scFv M Suzuki et al 783 Figure 3 Expression of scFv with charged tails in Pichia pastris. Three clones each of MutS (methanol utilization slow) and Mut+ (methanol utilization plus) bearing scFv with positively charged tails were cultured with 0.5% (v/v) methanol at 301C for 48 h. TCA-precipitates of medium were analyzed by immunostaining with anti-5 Â His antibody. MutS indicates a phenotype resulted from disruption of host AOXI gene by homologous recombination of vector, and Mut+ indicates a phenotype from nonhomologous integration. (À): Cells bearing scFv without charged tail. Figure 4 Effect of salt concentrations on expression of scFv with negatively charged tails in Pichia pastris. (a) MutS and Mut+ clones bearing scFv with negatively charged tails that had been adapted in 1.0 M NaCl were cultured at 301C for 48 h in YP medium with 0.5% (v/v) methanol containing 0, 0.5 and 1.0 M NaCl. Media were processed for immunostaining analysis with anti-5 Â His antibody. ( : scFv with negatively charged tail (D4S Â 5). (b) Three MutS clones bearing scFv gene with positively charged tails (K8S, R8S and R4S Â 2) were analyzed as shown in (a). ( : scFvs. Effect of salt on expression of recombinant antibody in which was confirmed by SDS-PAGE and silver staining yeast P. pastris (Figure 5a, b). The yield of scFv–D4S Â 5 was 5 mg/l of Interestingly, the secretion of recombinant antibody with culture with a purity of over 95%.
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