Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
1998-09-25
2003-09-16
Low, Christopher S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C530S351000, C530S395000, C530S399000, C530S402000, C530S324000, C435S041000, C435S068100, C514S002600, C514S008100, C514S012200
Reexamination Certificate
active
06620916
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a modified protein which has a liver accumulation property that its binding affinity for an asialoglycoprotein receptor present on a surface of a hepatocyte is lower than that of an asialoorosomucoid, and which can be produced by reacting a physiologically active protein containing one Gln residue that becomes a substrate for a transglutaminase with a branched-chain ligand composed of an amino acid derivative containing branched Gal or branched GalNAc in the presence of a transglutaminase to form an amide bond between the &ggr;-carboxyamide group of the glutamine residue in the physiologically active protein and the terminal primary amino group in the branched-chain ligand, a process for producing the same, and a pharmaceutical composition containing the above-mentioned protein.
The modified protein of the present invention is useful as an active ingredient of medications since the physiologically active protein used as a starting material retains a physiological activity.
TECHNICAL BACKGROUND
In recent years, as a result of the development of the biotechnology, a variety of physiologically active proteins can be mass-produced, and they have been expected to be candidate substances of new medications. However, there have been a large number of problems to be solved in order to put the same into practical use. Of these, the strict control of the pharmacokinetics thereof has been considered to be an important subject for increasing therapeutic effects and reducing side effects. For example, a great many investigations have been conducted on antitumor effects of recombinant human interleukin-2 (rhIL-2). The effects are identified with respect to mouse sarcoma and mammary tumor, and antitumor effects to melanoma and hemangioendothelioma are clinically identified. However, no expected effects to solid carcinoma of digestive organs are identified with rhIL-2 alone either in the animal test or clinically. Further, since rhIL-2 has a short half-life in the blood after the intravenous administration, a high dose thereof is required to exhibit the antitumor activity. Nevertheless, when rhIL-2 is administered at a high dose, a serious side effect called capillary leak syndrome occurs, providing an. influence such as an edema or the like in the lung or the liver. It is necessary to control the chemotherapeutic fate thereof for increasing the therapeutic effect of rhIL-2. In order to solve such a problem, investigations have been lately conducted with respect to an immunotherapy on metastatic and primary liver cancers by accumulating rhIL-2 around liver sinusoidal cells using IL-2 preparations of rhIL-2-containing liposomes or galactose-containing liposomes and increasing an activity of liver sinusoidal lymphocytes or the like (Jpn. J. Cancer Chemother., (1994), 21(13), 2105-2107).
Meanwhile, it is known that mammallian hepatocytes have an asialoglycoprotein receptor (hereinafter abbreviated as “ASGR”) which is a specific membrane-binding receptor to a glycoprotein having galactose (hereinafter abbreviated as “Gal”) or N-acetylgalactosamine (hereinafter abbreviated as “GalNAc”) in a branched sugar chain terminal (Ashwell, G., et al., Annu. Rev. Biochem., 1982, 51, 531-554). An uptake mechanism of the sugar protein with the receptor has a high binding affinity and is strong. In view of these properties and the specific presence of ASGR in hepatocytes, the above-mentioned receptor has attracted attention as a targeting system for specifically delivering medicines or genetic DNAs in metabolically important target cells (Wu, G.
Y. et al., J. Biol. Chem., (1987), 262, 4429-4432).
As a result of studies on the structure-activity interrelation of branched oligosaccharide chains or synthetic Gal derivatives isolated in the sugar recognition mechanism with the above-mentioned receptor, it has been clarified in view of the distance between Gal residues and the branching pattern that the intensity of the binding affinity is in the order of tetra-antenna type Gal>tri-antenna type Gal>bi-antenna type Gal>mono-antenna type Gal (Lee, Y. C. et al., J. Biol. Chem., (1983), 258, 199-202; Kawaguchi, K. et al., Arch. Biochem. Biophys., (1980), 205, 388-395; Connolly D. T. et al., J. Biol. Chem., (1982), 257, 939-945; Lee, R. T., et al., Biochemistry, (1984), 23, 4255-4259).
According to these findings, attempts have been made to accumulate DNAs or liposomes in the liver using a synthetic ligand containing branched Gal or GalNAc and to incorporate the same into cells (Japanese Laid-Open (Kokai) No. 202,085/1993; Haensler J. et al., Bioconjugate Chem., (1993), 4, 85-93; and Merwin, J. R., Bioconjugate Chem., (1994), 5, 612-620).
However, in the drug delivery system (hereinafter abbreviated as “DDS”) using an endocytosis mechanism through such a receptor, a low efficiency of intracellular uptake into the cells through a synthetic ligand is at issue. It has been made clear that this is because the binding affinity of such a synthetic ligand for ASGR is lower than a sugar chain of a glycoprotein such as asialoorosomucoid (ASOR), asialofetuin or the like which is a natural ligand [Lee, R. T. et al., Glycoconjugate J., (1987) 4, 317-328; and Biessen, E. A. L. et al., J. Med. Chem., 1995, 38, 1538-1546). Because of the low binding affinity, the synthetic ligand having Gal or GalNAc cannot be bound well to ASGR, and it is hard to incorporate ASGR into hepatocytes. Thus, the pharmaceutical effect is not exhibited well.
On the other hand, we have developed a method in which a protein is site-specifically modified with alkylamine derivatives of various compounds using an animal transglutaminase. Nevertheless, in this method, it is difficult that alkylamine derivatives are bound to a glutamine residue in an amino acid sequence of a physiologically active protein, for example, rhIL-2. Thus, a modified compound in which the alkylamine derivative is bound to the physiologically active protein derivative by introducing a peptide having a glutamine residue which is bindable using an animal transglutaminase into the physiologically active protein (PCT/JP 95/00298 (WO 96/06181)).
We have further investigated that the glutamine residue in the amino acid sequence of the physiologically active protein can be site-specifically modified using, among transglutaminases, especially a transglutaminase (B-TG) derived from microorganisms and having a wide substrate specificity to a Gln residue of a protein and using polylysine or alkylamine derivatives of an polyalkylene glycol such as polyethylene glycol without newly introducing a peptide molecule into an amino acid sequence of a physiologically active protein such as rhIL-2 or the like (Japanese Patent Application No. 270,102/1994 (Japanese Laid-Open (Kokai) No. 89,278/1996)); and PCT/JP 95/01994 (WO 96/10089)).
The present inventors have conducted various investigations to solve the problems that the compound modified with the synthetic ligand has a low affinity for ASGR and the modified compound is hardly incorporated into hepatocytes, and that for producing such a compound modified with the synthetic ligand, a peptide having a Gln residue has to be bound to a physiologically active protein.
The present inventors have attracted attention to the fact that if the physiologically active protein modified with such a synthetic ligand can directly act on cells other than hepatocytes, for examples, liver sinusoidal lymphocytes to exhibit the pharmaceutical effect of the physiologically active protein, the physiologically active proteins such as cytokine and the like are accumulated in the liver rather utilizing the low binding affinity for hepatocytes provided by such a synthetic ligand-modified compound, and that since the physiologically active substance is hardly incorporated into hepatocytes, it can be maintained in the liver, with the result that the activity of cytokine is expected to be exhibited by being bound to a specific receptor present on a cell membrane of the other target cell in the liver.
The targeting to the li
Ejima Chieko
Hayashi Eiko
Sato Haruya
Suzuki Manabu
Tabata Tomoyuki
Ajinomoto Co. Inc.
Low Christopher S. F.
Lukton David
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
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