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We have developed a novel methodology for site-specific pegylation of proteins by use of transglutaminase (TGase). In this methodology, alkylamine derivatives of poly(ethyleneglycol) (PEG) could be site-specifically incorporated into intact or chimeric proteins without decreasing the bioactivities. The incorporation site of the TGase-catalyzed modification is limited to the substrate Gln residues for TGases. The high homogeneity of the constructed conjugates and the ability to design conjugates with suitable incorporation sites will improve the applicability of PEG–protein conjugates for clinical use.

Purpose. To demonstrate the utilities of a synthetic low-affinity ligand ((Gal)3) for the asialoglycoprotein receptor (ASGP-R) as a hepatic targeting device for therapeutic cytokines.

To expand the applications of poly(ethylene glycol) (PEG)-protein conjugates for clinical use, we have developed a novel method for dual and site-specific incorporations of PEG derivatives into proteins using a substrate peptide (AQQIVM, named TG2) and transglutaminase (TGase). In our previous studies, TG2 was shown to be a special peptide with two adjacent Gln substrates for guinea pig liver transglutaminase (G-TGase). We have now constructed a chimeric protein (named rTG2-IL-2) of human interleukin-2 (IL-2), in which TG2 was fused to the N-terminus of IL-2. For the G-TGase-catalyzed reaction, rTG2-IL-2 was dually and site-specifically modified with alkylamine derivatives of PEG (PEG10, average M(r) 10 kDa) at both the Gln2 and Gln3 residues in the appended tag. To demonstrate the effectiveness of the G-TGase-catalyzed PEG-incorporation, we have compared the characteristics and the biological properties of PEG10-rTG2-IL-2 species with two PEG10 molecules attached to rTG2-IL-2 [(PEG10)(2)-rTG2-IL-2] with that of (PEG10)(2)-rhIL-2(R), in which PEG10 was randomly incorporated into rhIL-2 by a general procedure using a N-hydroxysuccinimidyl ester of PEG (PEG10-COOSu) (M(r) 10 kDa). (PEG10)(2)-rTG2-IL-2 was found to be superior in its in vitro bioactivities and equivalent in its pharmacokinetic profiles to (PEG10)(2)-rhIL-2(R). Unlike most previous methods, this approach can place dual PEG chains at designed sites on chimeric proteins without decreasing their bioactivities. Thus, TGase-catalyzed PEG-incorporation would improve the therapeutic utility of PEG-protein conjugates.

Reversed-phase HPLC (RP-HPLC) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) were used to characterize the transglutaminase (TGase)-catalyzed dual modification of a peptide (EAQQIVM, named FibN) with monodansylcadaverine (MDC). The synthesized FibN peptide, which was derived from the N-terminal sequence of fibronectin, was used as the substrate for a guinea pig liver TGase (G-TGase). The time course of incorporation of MDC into FibN, detected by RP-HPLC, indicated two separate fluorescent product peaks. ESI-MS analysis of the isolated fractions indicated that products represented MDC-incorporated FibN molecules in molar ratios of 1:1 ((MDC)–FibN) and 2:1 ((MDC) 2 –FibN). A sequence analysis of MDC–FibN, using ESI-MS/MS, showed that the first modified residue in FibN was mainly Gln3. The kinetic analysis of MDC incorporation suggested that dual incorporation would occur by mainly one route. A one-dimensional 1 H NMR comparison of MDC–FibN and unmodified FibN suggested that the first incorporation of MDC at Gln3 altered the substrate reactivity of the Gln4 residue in FibN for the G-TGase-catalyzed reaction. Thus, a detailed analysis of the peptide products using RP-HPLC and ESI-MS/MS should provide a powerful tool for exploring the mechanism of the substrate requirements of TGases.

Exploring a new method for the site-specific incorporation of functional groups into proteins, we have studied the combined use of genetic engineering techniques and enzymatic methods. Specifically, a short peptide for use as a substrate of guinea pig liver transglutaminase (TGase) is introduced at the N terminus of human interleukin-2 (hIL-2). The expressed chimeric protein (rTG1-IL-2) is chemically modified at a glutamine site in the appended sequence by TGase-catalyzed transamination with two amines, monodansylcadaverine (MDC), or a constructed derivative of poly (oxyethylene) (POE3). For the TGase-catalyzed modifications with MDC and POE3, 1 mol of donor was incorporated per mole of rTG1-IL-2, respectively. N-Terminal sequence analysis of MDC-modified rTG1-IL-2 (MDC-rTG1-IL-2) showed that the Gln-4 residue in the chimeric protein was site specifically modified with MDC. On the other hand, tryptic mapping of POE3-modified rTG1-IL-2 (POE3-rTG1-IL-2) by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOFMS) suggested that one of the Gln sites in the appended sequence was modified with POE3. The POE3-rTG1-IL-2 retained full bioactivity relative to the unmodified molecule and rhIL-2. This methodology could be a new and general route for the site-specific modification of proteins.

This paper proposes separated master system to decrease operational force of bilateral control in free motion. Operational force in bilateral control is not desirable because it prevent accurate reproduction of remote environmental force. Thus to decrease operational force is important. The master system consists of a grip part and an actuator part. A human operator manipulates the grip part. In free motion, both the actuator part of master robot and the slave robot track the grip part. With separating actuator, human operator feels fewer inertial force and friction when in free motion. When the slave robot contacts an object, the grip part contacts the actuator part and the operator feels reaction force from the object. The actuators tracked grip motion and the law of action and reaction was realized in experiment.

To demonstrate the utilities of a synthetic low-affinity ligand ((Gal)3) for the asialoglycoprotein receptor (ASGP-R) as a hepatic targeting device for therapeutic cytokines.The site-specific incorporation of (Gal)3 or a typical high-affinity ligand (GaINAc)3 into IL-2 was catalyzed by microbial transglutaminase. The anti-tumor activities, pharmacokinetic profiles and receptor-mediated endocytosis in hepatocytes of the ligand-IL-2 conjugates were examined in mouse.The (Gal)3 has approximately 50 times lower affinity to ASGP-R than (GalNAc)3. Nevertheless, the antitumor effects were in the order of (Gal)3-IL-2unmodified IL-2(GalNAc)3-IL-2. The systemic elimination and the hepatic uptake of (GalNAc)374L-2 were more rapid than (Gal)3-IL-2. The ratio of the rate constant representing dissociation from the cell-surface receptor (k(off) to that representing endocytosis of the ligand (k(int) was greater for (Gal)3-IL-2 than (GalNAc)s-IL-2, suggesting that (Gal)3-IL-2 preferably avoids internalization due to its lower affinity to the receptor. The simulation studies demonstrated that (Gal)3-L-2 was present in the hepatic extracellular space for a longer period than (GaINAc)3 IL-2.The (Gal)3 ligand increases the therapeutic efficacy of IL-2 by enhancing its exposure to the cell-surface. The k(off)/k(int) affects the targeting efficacy of the conjugates to ASGP-R.

A guinea pig liver transglutaminase (G-TGase)-mediated procedure for the site-specific modification of chimeric proteins was recently reported. Here, an alternative method with advantages over the recent approach is described. This protocol utilizes a microbial transglutaminase (M-TGase) instead of the G-TGase as the catalyst. M-TGase, which has rather broad structural requirements as compared to the G-TGase, tends to catalyze an acyl transfer reaction between the gamma-carboxamide group of a intact protein-bound glutamine residue and various primary amines. To demonstrate the applicability of the M-TGase-catalyzed protein modification in a drug delivery system, we have utilized recombinant human interleukin 2 (rhIL-2) as the target protein and two synthetic alkylamine derivatives of poly(ethyleneglycol) (PEG12; MW 12 kDa) and galactose-terminated triantennary glycosides ((Gal)(3))) as the modifiers. For the M-TGase-catalyzed reaction with PEG12 and (Gal)(3), 1 mol of alkylamine was incorporated per mole of rhIL-2, respectively. Peptide mapping of (Gal)(3)-modified rhIL-2 ((Gal)(3)-rhIL-2) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) suggested that the Gln74 residue in rhIL-2 was site specifically modified with (Gal)(3). The PEG12-rhIL-2 and (Gal)(3)-rhIL-2 conjugates retained full bioactivity relative to the unmodified rhIL-2. In pharmacokinetic studies, PEG12-rhIL-2 was eliminated more slowly from the circulation than rhIL-2, whereas (Gal)(3)-rhIL-2 accumulated in the liver via hepatic asialoglycoprotein receptor binding. The results of this study expand the applicability of the TGase-catalyzed methodology for the preparation of protein conjugates for clinical use.

To develop a receptor-mediated gene delivery system into hepatoma cells using the cationic alpha-helical peptide as the gene carrier molecule, we modified an alpha-helical peptide, which is known to have transfection abilities into cells, with a multi-antennary ligand containing several galactose residues that provide efficient binding to the asialoglycoprotein receptor. The galactose-modified peptides formed complexes with a plasmid DNA and showed gene transfer abilities into HuH-7 cells, a human hepatoma cell line. The transfection efficiency of the peptide was increased by increasing the number of modified galactose residues on the peptide. Furthermore, considerable inhibition of the transfection efficiency by the addition of asialofetuin, which is a ligand for the asialoglycoprotein receptor, was observed in all galactose-modified peptides. Based on this result, we could confirm that the internalization of the galactose-modified peptides occurred by the receptor-mediated endocytosis pathway. In addition, to understand the transport route of the peptide-DNA complex in the cell, the effects on the transfection efficiencies with several endocytosis inhibitors were examined. As a result, it was suggested that the translocation of the peptide-DNA complex from the endocytic compartments to the cytosol mainly occurred during an early endosome step.

A conjugate of Cu,Zn-superoxide dismutase (SOD) with a strong iron chelating agent, deferoxamine (DFO), was synthesized (SOD-POE-DFO) via polyoxyethylene (POE) as a linking agent. N-terminal amino groups of lysine residues in SOD are modified with 1:1 binding products of polyoxyethylene and deferoxamine (POE-DFO) through a covalent amido bond. The mean number of the POE-DFO bound per one SOD molecule is calculated to be 3.3 by determining the C/N ratio after elemental analysis. The half-life of the SOD-POE-DFO is about 1.2 h in rats, whereas that of free SOD is about 5-10 min. POE plays the part not only of the linking agent but also of expanding the lifetime in the circulation. The SOD-POE-DFO possesses both the metal chelating ability (for DFO) and the ability of scavenging superoxide radicals (for SOD). Therefore, the SOD-POE-DFO of the present study can eliminate the superoxide radical and free iron simultaneously and in the same location, and thus, it would be a molecular device with multiple functions which prevents the damage to tissues by scavenging the variety of reactive oxygen species.