View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Kidney International, VoL 47 (1995), pp.1327—1335 Renal accumulation of streptavidin: Potential use for targeted therapy to the kidney BILu SCHECHTER, RUTH ARNON, CHRISTOPHE COLAS, TATYANA BURAKOVA, and MEIR WILCHEK Departments of Chemical Immunology and Membrane Research & Biophysics, The Weizmann Institute of Science, Rehovot, Israel Renal accumulation of streptavidin: Potential use for targeted therapyeach other, as evidenced by their lack of immunological cross- to the kidney. Streptavidin exhibits a remarkable accumulation in the reactivity, remote evolutionary origin and differences in charge kidney. Biodistribution studies with radio-iodinated streptavidin showed that 70 to 80% of the injected dose per gram tissue (%/g) were retained and glycosylation. Despite some similarity in a series of short in kidneys of Balb/C mice for three to four days compared to less than interrupted segments, the amino acid composition and primary 5 %/g levels in other tissues. This observation means that 15 to 20% of the sequence are also different [11]. The comparison between these injected dose is accumulated in the kidney, an organ that constitutes less two proteins has recently been extended by us to include their in than 1% of total body weight. Similar results of percent radioactivity pervivo distribution pattern upon intravenous (i.v.) administration total kidney were obtained in other mouse strains as well as in rats and rabbits. Avidin, or the post-secretory form of streptavidin which is of a [12]. Biodistribution studies performed with radio-iodinated avi- higher molecular weight, do not show any preferential affinity to thedin showed a relatively rapid clearance, with only residual levels in kidney. The kidney-accumulated streptavidin was mostly confined to the blood and most organs, including the kidney, already at six hours cortex, concentrated in the proximal tubular cells. Accumulation ofafter injection. streptavidin in the kidney was independent of biotin, since addition of biotin to radio-iodinated streptavidin prior to injection did not affect its Biodistribution of streptavidin was found to be dependent on a kidney uptake. Therefore, streptavidin, which aquires its kidney accumu- truncation process occurring during its purification. Recent stud- lation property following truncation of the native form, may be utilized for ies have shown that the post-secretory form of streptavidin is renal specific delivery of chemotherapeutic agents, radioactive isotopes larger than the currently used form. Upon certain isolation and other effector molecules. Such ligands can be linked to streptavidin via procedures the 18 kDa subunit of the post-secretory streptavidin conventional coupling methods or following their biotinylation. Prelimi- nary experiments showed that streptavidin can target to the kidneymay undergo proteolytic degradation to give a truncated form of biotinylated ligands or high doses of chemically linked radionuclides. a molecular size of about 14 kDa [13]. The truncation is effected through the cleavage of 12 to 14 amino acid residues at the N-terminal and up to 18 residues at the C-terminal end of each subunit. Whereas the 18 kDa streptavidin subunit was found to be Streptavidin is a 58 kDa non-glycosylated neutral protein ofsensitive to the action of several commercially available proteo- Streptomyces avidinii, which displays a remarkably strong bindinglytic enzymes, the truncated form exhibits a remarkable resistance affinity (1015 M') for the vitamin biotin [1]. This protein is similarto any proteolytic activity [14]. Biodistribution studies in mice in structure and biotin-binding properties to its counterpart,performed with the intact post-secretory form of streptavidin avidin, a positively charged egg-white glycoprotein [2]. Bothshowed a normal rate of protein clearance, with levels of 20%/g in proteins are tetramers containing one biotin-binding site perblood at six hours and 2 to 12%/g in blood and tissues at 24 hr (a subunit. The streptavidin- or avidin-biotin complexes have pro-pattern similar to most proteins). On the other hand, the trun- vided extremely useful and versatile intermediates in a variety ofcated form of streptavidin (most commercial preparations) had biological and analytical systems [3, 4]. Avidin or streptavidin,low blood and tissue levels, but its kidney uptake was exception- either in their native form, or when coupled to various reporterally high [12]. probes such as fluorescent dyes, radioactive elements, enzymes or In the present study we extended the investigation on the immobilized matrices, can recognize biotin coupled to various lowselective uptake of streptavidin (the commercial truncated form) or high molecular weight substances. More recently, the use ofby the kidney, referring to parameters such as the effect of time on these two systems has been extended to in vivo procedures forthe accumulation and clearance patterns of streptavidin, occur- cancer diagnosis and treatment such as radioimmunodetectionrence of this phenomenon in other animal species, localization [5—8]anddrug immunotargeting [9, 10]. Radiolabeled or drug-region of streptavidin in the kidney, effect of biotin on kidney bound avidin could be targeted to biotinylated antibodies concen-uptake, and the potential use of streptavidin as a targeting device trated at the tumor site. to the kidney. Nonetheless, avidin and streptavidin are distinct and differ from Methods Received for publication September 14, 1994 and in revised form December 5, 1994 Materials. Post-secretory streptavidin, provided by E. Bayer and Accepted for publication December 5, 1994 H. Ben-Hur, was purified from the spent culture broth of Strep- © 1995 by the International Society of Nephrology tomyces avidinii using a modified iminobiotin-containing affinity 1327 1328 Schechteret al: Streptavidin accumulation in the kidney 100 80 C) U) 60 C) ()C) 00 40 0C) C) Fig. 1. Biodistribution of '251-streptavidin. C 20 Comparison between native (intact, i.v.) and truncated (iv. and i.p.) streptavidin at 24 hours following injection was performed in pairs of _RI_.., Balb/C mice (1 to 2 j.g protein, 1 sCi/mouse). 0'- Data are mean percent of injected radioactivity KS LHLuMBBI- KS LHLuMBBI- KS L H LuM B BI dose/gram tissue so.Abbreviations are: K, kidney; S, spleen; L, liver; H, heart; Lu, lung; Native (iv.) Truncated (iv.) Truncated (i.p.) M, muscle; B, bone; BI, blood. 80 60 a) U) U) C) C) 0U) C) 0 C) C 20 —Ill——I II— —II. — — — 0 .Iul_.I 1111._I"I——I I KS LH LuMBB1-KS LH LuMBB1-K S LH LuMBB1-KS LH LuMBB1-KS LH LuMBB1-KS LH —K S LH -K S LH 2hr 5hr 8hr l2hr 24hr 48hr 72hr 86hr Fig. 2.Biodistrihutionof '251.streptavidin (truncated) at different time intervals following iv. injection into pairs of BalbIc mice. Legends and data are as in Figure 1. column [13]. Streptavidin was provided as a gift by Boehringer- Radiolabeling. Radiolabeling of tyrosine residues in proteins Mannheim GmbH (Mannheim, Germany). Biotinyl-tyrosine with 1251or131jwasperformed by the Chloramine-T method [15]. (b-Y) was prepared by reacting biotin-N-hydroxysuccinimide (355Protein (0.2 ml of I mg/mI) in 0.02 M sodium phosphate buffer mg) dissolved in 2 ml DMF with tyrosine (269 mg) in 2 ml of 1 MpH 8 was reacted with 0.5 mCi or 2.5 mCi of Na1251 or Na'31 I in NaOH.After 24 hours at room temperature the solvents werethe presence of Chloramine-T (10 pJ of 2 mg/ml). After two evaporated and the b-Y was radioiodinated. Biotinyl-alanyl-ala-minutes at room temperature the reaction was terminated by nyl-tyrosine (b-AAY) was similarly prepared by reacting biotin-adding sodium metabisulfite (10 sl of 2 mg/mI). The radiolabeled N-hydroxysuccinimide with AAY (the same molar ratio). Biotinyl- proteins were purified by gel exclusion chromatography on cs-melanotropin hormone (b-aa-MSH) was provided by Dr. Y.Sepharose G-25 (Pharmacia Fine Chemicals, Piscataway, NJ; Salomon. Radioactive iodine, 25Ior131Jwaspurchased fromUSA). Radio-iodinated b-Y, b-AAY (20 tg) and b-a-MSH (1.5 Amersham International plc. (Buckinghamshire UK). mg) similarly prepared were complexed to 0.2 mg streptavidin (30 Schechter et al: Streptavidin accumulation in the kidney 1329 A C (0 C, 0 a, t0 t0 0) 0 C) C B 80 20 3. a 60 15 U) (I) 2 Dl 10 4o0 0 Fig.3. Quantitative retention of '251-streptavidin -c a) 1 in whole organs (A)orbiodistribution of 125j oC) 20 5. streptavidin(B)at24 hours following injection C into a Balb/c mouse, Wistar rat and New Zealand rabbit. Data are expressed as percent of injected radioactivity dose/organ (A) or /gram tissue (B). Symbols are: (U), kidney; (P4) spleen, (L) 0 liver; (B) heart; (LI) lung; (LI) muscle; (B) Mouse Rat Rabbit blood. mm at room temperature) priOr to purification on Sepharose 0-25. 120 50 ioo :i Biodistribution and pre-targeting studies U) 40a) Ci) 80 C In biodistribution experiments, age- (10 to 12 weeks), sex- and 0) 30B weight-matched Balb/c mice were given i.v. injections, into the 60 lateral tail vein, of the radio-iodinated proteins or radio-iodinated 40 20 biotinyl-derivatives complexed to streptavidin (1 j.tCi/mouse). At time intervals as indicated below, blood samples were withdrawn 20 10 from the tail, mice were killed and organs were dissected out. 0 0 Organs were washed in phosphate buffered saline (PBS), blotted 0 100 200 300 400 dry, weighed and counted for radioactivity. The results are Streptavidininjected, tg expressed as mean percentage of injected radioactivity dose per orper whole organ. Biodistribution of 1251.. Fig.4. Biodistriburion of a constant amount of '251-streptavidin (at 24 h) gram tissue (%/g), mixed with increasing amounts of cold streptavidin prior to injection into a streptavidinin a rat or a rabbit was performed following injectionBalblc mouse. Data are expressed as percent of injected radioactivity into the tail or ear vein, respectively, of radio-iodinated strepta-dose/gram tissue or as g streptavidin/kidney.