Molecular Diversity, 8: 101–111, 2004. KLUWER/ESCOM © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 101 Full-length paper New transport peptides broaden the horizon of applications for peptidic pharmaceuticals J. P. M. Langedijk 1,∗ , T. Olijhoek 1 , D. Schut 1 , R. Autar 1 & R. H. Meloen 1,2 1 Pepscan Systems B.V., Lelystad, The Netherlands; 2 Academic Biomedical Centre, Yalelaan 1, University Utrecht, The Netherlands ( ∗ Author for correspondence, E-mail: j.p.m.langedijk@pepscan.nl) Received 10 August 2003; Accepted 4 October 2003 Key words: CPP, E rns , protein transduction, PTD, Tat, transport peptide Summary Protein transduction domains (PTDs) have proven to be an invaluable tool to transduce a wide variety of cargo’s including peptides across the plasma membrane and into intact tissue. The PTDs are able to deliver biologically active molecules both in vitro and in vivo. This study describes many new polybasic PTDs of which some are just as potent as the PTDs derived from extracellular RNAses or other published PTDs. Large differences in potency became apparent when the PTDs are coupled to particular cargoes. Therefore, the unique characteristic of a PTD may only become apparent when it is selected for a particular application. Rules for optimization of PTDs for particular applications are now emerging and open the way for a new generation of drug delivery agents. Because fixation artifacts and irreversible membrane binding may cause misinterpretation of the amount of internalization of polybasic peptides, we have developed an enzyme transduction assay based on the intracellular loading of a cell permeable substrate. In this assay, a fluorescent signal is generated by internalized enzyme in intact cells and not by membrane-bound or extracellular enzyme. Introduction A major challenge in today’s biotechnological drug devel- opment is the fact that many recombinant products such as bioactive proteins, enzymes, antibodies, peptides, antis- ense oligonucleotides and gene constructs, have inherently unfavorable pharmacokinetic properties due to their physi- cochemical nature. None of these molecules have the ability to readily penetrate cell membranes and the body’s physiolo- gical barriers to reach their pharmacological targets. This usually leads to low in vivo ‘effective availability’, that is the amount of drug that eventually reaches its site of action, and makes otherwise promising molecules such as peptides unsuitable for drug therapy. In the past few years, a variety of peptides were shown to be capable of crossing biological membranes of a variety of cell types [1, 2]. The use of such peptides for delivery of biomolecular cargoes, such as proteins and peptides, into living cells has been emerging as a novel methodology for cellular biology and drug delivery. Among these so-called transport peptides, or protein transduction domains (PTDs), the HIV-1 derived Tat pep- tide and the penetratin peptide derived from Antennapedia homeobox protein are regarded as representative [3, 4]. The PTDs derived from extracellular RNAses that we have re- cently described are part of this growing family of transport peptides [5]. Transport peptides or PTDs are generally short cationic peptides that possess the ability to traverse the lipid bilayer of cells in a concentration-dependent manner that is probably independent of specific receptors or transporters. Most PTDs that have been described until now are short peptide sequences corresponding to a short stretch of the original native protein that have a function in binding poly- anionic regions like RNA, DNA or sulfated carbohydrates like heparin. This inherent trait of PTDs to bind to poly- anions may facilitate several different steps of the journey of the extracellular RNase or other protein cargoes to it’s intracellular target: binding heparan sulfate proteoglycan (HSPG) in the extracellular matrix to catch the PTD and concentrate it close to the cell surface [6, 7], binding the negatively charged headgroups of phospholipids at the mem- brane surface [8], disturbing the plasma or endosomal mem- brane and finally binding a polyanionic substrate. For most PTDs the biological relevance for the translocation activity could not be proven. In fact, it is likely that the stretch of basic residues may have another function and happens to be able to leak into cells unintentionally [9]. Such PTDs could be considered ‘fortuitous’. The PTDs derived from the extracellular RNAses that we have described may form an exception. To hydrolyze their target RNA, these RNAses