Adapter Protein for Site-Specific Conjugation of Payloads for Targeted Drug Delivery Marina V. Backer,* ,† Timur I. Gaynutdinov, † Vimal Patel, † Brian T. Jehning, † Eugene Myshkin, ‡ and Joseph M. Backer † Rammelkamp Center for Research, Case Western Reserve University School of Medicine, Cleveland, Ohio 44109, and SibTech, Inc., Newington, Connecticut 06111. Received March 3, 2004; Revised Manuscript Received July 21, 2004 High-affinity interactions of two fragments of human RNase I (1-15-aa Hu-tag and 21-125-aa HuS adapter protein) can be used for assembly of targeting drug delivery complexes. In this approach, a targeting protein is expressed as a fusion protein with a 15-aa Hu-tag, while HuS is conjugated to a drug (or a drug carrier) creating a “payload” module, which is then bound noncovalently to the Hu- tag of the targeting protein. Although this approach eliminates chemical modifications of targeting proteins, the payload modules are still constructed by random cross-linking of drugs or drug carriers to an adapter protein that might lead to functional heterogeneity of the complexes. To avoid this problem, we engineered an adapter protein HuS(N88C) with an unpaired cysteine in position 88 that can be directly modified without interference with activity of assembled targeting complexes. HuS- (N88C) binds Hu-tagged annexin V with K D of 50 ( 6 nM, which is comparable to that of wild-type HuS. To demonstrate the utility of HuS(N88C) for developing uniform payload modules, we constructed a HuS(N88C)-lipid conjugate and inserted it into preformed liposomes loaded with a fluorescent dye. Targeting proteins, Hu-tagged vascular endothelial growth factor or Hu-tagged annexin V, were docked to liposomes decorated with HuS, and the assembled complexes delivered liposomes selectively to target cells. INTRODUCTION Efficient technology for loading drugs or drug carriers onto targeting proteins is necessary for targeted drug delivery (1). Currently, loading is largely based on random chemical conjugation of a cargo, such as drugs, drug carriers, or adapters for drug carriers, directly to targeting proteins. Chemical modifications of targeting proteins often damage their ability to bind to cellular targets, require expensive custom development, and yield heterogeneous preparations. To solve these problems, we have recently proposed to link a cargo to a standardized adapter protein that binds noncovalently to a docking tag engineered into a targeting protein (2). Our current adapter/docking tag pair is based on interactions between two fragments of human RNase I (3). As an adapter protein, we are using a 21-127-amino acid (aa) fragment of human RNase I, named HuS, while its N-terminal 1-15-aa fragment fused to a targeting protein serves as a docking tag, named Hu-tag. In the first application of this approach in vivo, we imaged a small mouse tumor using assembled complexes of Tc 99m -radiolabeled HuS and Hu-tagged vascular endothelial growth factor (4). Although this approach eliminates chemical modifica- tions of targeting proteins, assembled complexes are still structurally (and, potentially, functionally) heterogeneous because an adapter protein is still loaded via random cross-linking. Furthermore, random chemical modifica- tions of adapter protein, particularly with large cargos such as liposomes or nanoparticles, decrease a proportion of molecules retaining full tag-binding activity. To solve these problems, we engineered an adapter protein with an unpaired cysteine in position 88, HuS(N88C), that can be directly modified without interference with activ- ity of assembled targeting complexes. In this work, a PEGylated lipid was cross-linked to C88 in HuS(N88C), and the resulting conjugate was used to decorate lipo- somes (Figure 1). Hu-tagged vascular endothelial growth factor, or Hu-tagged annexin V, was docked to HuS- liposomes, and assembled complexes delivered liposomes selectively to target cells. MATERIALS AND METHODS Construction and Purification of Chimeric BH- RNase Mutants. Construction of chimeric 1-29 B/ 30-127 H- RNase (BH-RNase) consisting of a 1-29 aa fragment of bovine RNase A and a 30-127 aa fragment of human RNase I has been described recently (5). The N88C and G68C amino acid substitutions were introduced sepa- rately in the pET29/ 1-29 B/ 30-127 H-RNase plasmid DNA by site-directed mutagenesis (Gene-Tailor site-directed mu- tagenesis kit, Invitrogen, Carlsbad, CA). The following primers were used for mutagenesis: 5′-TC ACT GAC T- GC CGT CTT ACT TGC GGA TCC CGT T (sense, muta- tion underlined) and 5′-AGT AAG ACG GCA GTC AGT- GAT ATG CAT AGA A (antisense) for the N88C mutation and 5′-AG AAG GTT ACC TGC AAA AAT TGC CAG GG- T AAC TG (sense, mutation underlined) and 5′-ATT TTT GCA GGT AAC CTT CTC TTG GAA GCA A (antisense) for the G68C mutation. Both mutations were confirmed by sequencing. BH-RNase(G68C) and BH-RNase(N88C) were expressed in BL21(DE3) Escherichia coli (Novagen, * To whom correspondence should be addressed. Mailing address: SibTech, Inc., 705 North Mountain Road, Newington, CT 06111. Phone: (860) 953-1753. Fax: (860) 953-1317. E- mail: mbacker@sibtech.com. † SibTech, Inc. ‡ Case Western Reserve University School of Medicine. 1021 Bioconjugate Chem. 2004, 15, 1021-1029 10.1021/bc0499477 CCC: $27.50 © 2004 American Chemical Society Published on Web 08/24/2004