Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Derived from – or present in – food product
Reexamination Certificate
1998-06-19
2003-10-28
Kunz, Gary (Department: 1647)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Derived from, or present in, food product
C424S009362, C424S001690, C514S002600
Reexamination Certificate
active
06638508
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel derivatives of avidin-type molecules, particularly to avidin-type molecules modified with 2,4,6-trinitrophenyl (TNP) or lactosyl (Lac) groups, to antigen-antibody complexes of avidin-type molecules, and to compositions comprising them for targeted diagnostic and therapeutic treatment of the liver and the reticuloendothelial system.
BACKGROUND OF THE INVENTION
Drug delivery, using targeting systems wherein a drug or radioactive isotope are coupled to a specific targeting vehicle, has been studied for both therapeutic and diagnostic potentials. For the treatment of tumors, for example, targeting of drugs has been carried out via antibodies where the drug-antibody immunoconjugate is expected to localize specifically at a particular tumor cell. Limited specific uptake of immunoconjugates by human solid tumors was found to be the major limitation to drug or isotope immunotargeting, and therefore alternative vehicles for drug delivery other than antibodies showing specific affinity to a given tissue or organ, have been subject of further studies, such as, for example asialoglycoproteins for targeted delivery of small or large molecules to hepatocytes (Wu and Wu, 1993).
Streptavidin, a 52-60 kDa tetrameric non-glycosylated neutral protein which is a truncated form of the native streptavidin of
Streptomyces avidinii,
carries one biotin-binding site per monomer with a remarkably strong binding affinity to biotin (Chaiet and Wolf, 1964). The native, post-secretory form of streptavidin is larger and has a MW of 72 kDa (18 kDa per subunit). This native streptavidin molecule degrades rapidly to the stable 52-60 kDa streptavidin and can only be identified under special conditions. Bayer et al. (1989) reported that the native streptavidin undergoes proteolytic degradation during isolation to a truncated form with a molecular size of about 14 kDa per subunit, which is the commercial 52-60 kDa form of streptavidin known and recognized in the art. The truncation is effected through the cleavage of 12-14 amino acid residues at the N-terminal and up to 18 residues at the C-terminal end of each subunit. The 18 kDa streptavidin subunit was found to be sensitive to the action of several commercially available proteolytic enzymes, but once streptavidin (truncated form of the native molecule) is formed, it is remarkably stable to proteolytic activity (Wilchek and Bayer, 1989).
Streptavidin is similar in structure and biotin-binding properties to its counterpart avidin, a positively charged egg-white glycoprotein. The biotin-binding affinity of these two proteins is the highest recorded for any protein-ligand interaction (10
15
M
−1
). Both proteins are tetramers containing one biotin-binding site per subunit and have similarity in a series of short interrupted segments (Green, 1975; Argarana et al., 1986), but they differ from each other in charge and glycosylation as well as in general primary sequence. Resistance to proteolytic enzymes is shared by both streptavidin and native avidin.
The streptavidin- and avidin-biotin complexes have provided extremely useful and versatile intermediates in a variety of biological and analytical systems (Wilchek and Bayer, 1984; Wilchek and Bayer, 1988). In principle, biotin coupled to a large variety of molecules can be recognized by avidin or streptavidin, either in their unmodified form or when coupled to various reporter probes, such as fluorescent dyes, radioactive elements, enzymes or immobilized matrices. Later, the use of these two systems has been extended to include different in vivo procedures, such as radioimmunodetection (Hnatowich et al., 1987) and immunotargeting (Longman et al., 1995). The present inventors have previously shown indirect immunotargeting of cisplatin to human epidermoid carcinoma using the avidin-biotin system (Schechter et al., 1991).
Biodistribution studies in mice comparing radioiodinated (
125
I)-avidin, (
125
I)-native streptavidin and (
125
I)-streptavidin showed that, at 24 h, native streptavidin had a normal clearance pattern from all organs with retention levels of 1-10% of total injected dose per gram tissue (%/g) whereas avidin was cleared at a faster rate and was in the range of 0.2-3%/g (Schechter et al., 1990). In contrast, streptavidin exhibited a remarkable and prolonged accumulation in the kidney with uptake levels of 70-80% of the injected dose/g tissue (%/g), mostly confined to the kidney cortex, for a period of 3-4 days following i.v. or i.p. injection, whereas its levels in other organs was low (0.3-4%/g) (Schechter et al., 1995). In terms of organ accumulation, 15% of the total injected dose of streptavidin was accumulated in each kidney, an organ comprising only 1% of the total body weight. Similar results of organ accumulation were also obtained for rats and rabbits (Schechter et al., 1995).
Addition of exogenous biotin did not reduce kidney uptake and did not affect streptavidin biodistribution to other organs (Schechter et al., 1990), excluding the possibility that streptavidin accumulation occurs due to interaction in the kidney with free biotin or with biotinylated proteins. The observation that native streptavidin and avidin, both displaying biotin-binding affinity, did not accumulate in the kidney, also excludes the possibility that biotin or biotinyl groups in this organ serve as the major anchor for streptavidin accumulation.
Avidins are enzyme resistant carriers (Hiller et al., 1991; Ellison et al., 1995) that can serve to provide selective and prolonged organ accumulation to ensure prolonged maintenance of these carriers in the target organ. Streptavidin itself (52-60 kDa) is accumulated in the proximal tubule of the mouse kidney for 3-4 days. This is due to processing of low MW proteins (<64 kDa) which generally undergo tubular endocytosis and active lysosomal degradation. The exceptional long-term sequestration of streptavidin in the kidney is attributed to its unique resistance to enzymatic degradation (avidin, which is of a higher molecular size, 67 kDa, did not accumulate in the kidney and was rapidly cleared from the circulation and tissues).
Chemical modification of macromolecules can change the in vivo disposition profile of these macromolecules and lead to receptor-mediated targeting or other types of cellular uptake in the target organ targeted by these potential macromolecular homing devices. Hepatotropic markers which are receptor specific to terminal &bgr;-D-galactose or N-acetyl-&agr;-D-galactosamine present on mammalian parenchymal cells (hepatocytes) have been reported earlier (Ashwell and Hartford, 1982; Schwartz, 1984). The high affinity interaction with this receptor triggers efficient internalization of circulating asialoglycoproteins (ASGP), synthetic glycosylated proteins, or other macromolecules (neoglycoproteins or neoglycoconjugates) modified with saccharides (Lee and Lee, 1994). Thus, carbohydrate receptor-mediated targeting to parenchymal (via terminal &bgr;-D-galactose or N-acetyl-&agr;-D-galactosamine) and non-parenchymal (via terminal N-acetylglucosamine or mannose; Stahl and Schlesinger, 1989; Magnusson and Berg, 1989) cells of the liver has shown great promise as a potential delivery method using receptor-mediated endocytosis. The advantages of this system arise from the high affinity of the receptor for the ligand and the rapid recycling of the receptor molecule.
Natural asialoglycoproteins (ASGP), such as asialoorosomucoid and asialofetuin, were employed first, but later on, synthetic glycosylated proteins (neoglycoproteins) were used as prototypes of carrier systems. The rapid clearance of these carriers yielded attempts to slow down their degradation in order to achieve gradual but predominant accumulation in the target tissue. One of the approaches used chemical modification with biologically inert macromolecules, such as polyethyleneglycol (PEG). However, PEG conjugation was found to result in prolonged plasma retention due to reduction of interaction with tissues (Civitico, 1990; Crance, 1
Arnon Ruth
Schechter Bilha
Wilchek Meir
Browdy and Neimark PLLC
Hayes Robert C.
Kunz Gary
Yeda Research and Development Co. Ltd.
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