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Kidney International, VoL 47 (1995), pp.1327—1335

Renal accumulation of streptavidin: Potential use for targeted therapy to the kidney

BILu SCHECHTER, , CHRISTOPHE COLAS, TATYANA BURAKOVA, and MEIR WILCHEK

Departments of Chemical Immunology and Membrane Research & Biophysics, The Weizmann Institute of Science, ,

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. , 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 , 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 . 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 , 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 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. Symbols are: (0)radioac- vidin (6 to 7 Ci/animal). After 24 hours, blood was withdrawn,tivity dose; (X) streptavidin. the animals were killed, and tissues and organs were analyzed as described above. In experiments designed to evaluate the capacity of streptavidinarations were used as control. External radioactivity was moni- to target therapeutic doses of 1251and1311athymicnude micetored daily at a distance of 5 mm from the kidney or head region. were injected with the radio-iodinated derivatives (0.1 mCi!The results are expressed as the mean percentage of the decay in mouse). 125jand'311-labeled bovine serum albumin (BSA) prep-radioactivity in comparison to day 0. 1330 Schechteret al: Streptavidin accumulation in the kidney

AMouse B Rabbit 120 4

100 3 a) 80

60 2- V V a) '1)

1 20 Fig. 5. Biodistribution of '251-streptavidin in total kidney (K), medulla (M) and cortex (C). Data 0 0— are expressed as percent of injected KM C radioactivity dose/gram tissue for a rabbit (B) KMC KMC KMC KMC and mean percent of injected radioactivity dose/ l2hr 24hr 48hr 72hr 24 hr gram tissue su for 3 mice (A).

Immunohistochemical staining organs, including the kidney, was in the range of 2 to 10 %/g. Mice were injected i.v. with 500 xg streptavidin and the kidneysLevels of the truncated streptavidin in blood and most organs were removed 24 hours later. Staining was performed at roomwere lower, with radioactivity values that did not exceed 4%/g, temperature on 3 x frozen sections mounted on polylysine-coatedexcept for the kidney. Accumulation in the kidney was exception- glass slides, fixed with 4% buffered formaldehyde (20 mm) andally high after both intravenous and intraperitoneal (i.p.) injec- rinsed with PBS. The sections were then treated with 5% BSAJtions, with uptake levels ranging between 70 to 80%/g (around PBS (15 mm) and anti-streptavidin was applied (1 hr).15% per kidney). Addition of exogenous biotin did not affect the Endogenous peroxidase activity was blocked by incubating theaccumulation pattern of truncated streptavidin (data not shown). tissue with 3% H202 in ethanol (40 mm). The rinsed sections wereKidney accumulation and clearance of radioactivity as a function incubated with peroxidase-labeled goat-anti rabbit antibody (Bio-of time (Fig. 2) showed that already at two hours following i.v. Makor, Rehovot, Israel) at 1:100 dilution (1 hr) and peroxidaseinjection the level of radioactivity in the kidney was 22%/g, was revealed with 0.02% 3-amino-9-ethylcarbazole (Bio-Makor)increasing to more than 60%/g at 12 hours, peaking to 70%/g at 24 and 0.02% H202 in 20 m acetate buffer (pH 5.2). The sectionshours, and declining to 26%/g only at 86 hours. Radioactivity in were counterstained with Mayer's hematoxylin and mounted inspleen, liver, heart, lung, muscle and bone did not exceed 6%/g Kaiser's glycerol-gelatin (Sigma Diagnostics, St. Louis, MO,along this time period, except for blood radioactivity which was USA). In control staining immune antibody was replaced by a13%/g at two hours but dropped to 2%/g after 24 hours. Biodis- non-specific antibody. tribution experiments in rats and rabbits showed preferential Antibody. Anti-streptavidin antibody was prepared by multi-accumulation of streptavidin in the kidneys of these animal focal intra-dermal immunizations of rabbits with 3 X 1 mgspecies as well. Organ radioactivity values were similar to those streptavidin in complete Freund's adjuvant. The Ig fractions ofobserved in mice and ranged between 13 and 17% per kidney (Fig. preimmune (control) and immune sera were used at 50 xg/ml.3A). However, %/g levels were lower due to the larger size of the Autoradiography of streptavidin-containing frozen rat kidneyorgans, (Fig. 3B). Radioactivity levels in spleen, liver, heart, lung, sections (5 jx)wasperformed with kidneys obtained from rats 24muscle and blood were low so that kidney/other tissue ratios, as hours following the injection of 6 dxCi 25I-streptavidin. Thewell as total kidney uptake in the three animal species were sections were mounted on glass slides, dried for 30 minutes and comparable. apposed to radiation sensitive film (Hyperfilm-/3max, Amersham) The total amounts of streptavidin injected in the experiments for one day. The film was developed using standard methods. described above were in the range of 0.5 to 2.0 xg protein per Results mouse. A quantitative analysis of the kidney capacity to accumu- late streptavidin was carried out by injecting a constant amount of Biodistribution of streptavidin radioalabeled streptavidin mixed with increasing quantities of the A comparison of the biodistribution of intact and truncatedcold protein into mice. Assuming that radioactive and cold streptavidin at 24 hours following i.v. injection of the radioiodi-streptavidin do not differ in their kidney accumulation properties, nated proteins into Balb/c mice is illustrated in Figure 1. Asthe results presented in Figure 4 indicate that whereas 100 or 200 shown, radioactivity levels of intact streptavidin in blood andg cold streptavidin did not significantly affect '251-streptavidin Schechter et at: Streptavidin accumulation in the kidney 1331 J _ __Sc. ___- a. Ii 0

Fig.6. Immunohistochemical (immunoperoxidase) staining of streptavidin in kidney sections. Mouse kidneys were removed 24 hours following streptavidin injection (500 g) and sections were stained with anti-streptavidin polyclonal antibody (immune Ig). A. Staining of streptavidin sequestered in proximal tubules near capsule (indicated by arrows in A and C) using anti-streptavidin immune 1g. B. Negative staining of tubules with control (pre-immune) 1g. C. Positive staining of tubules near the glomeruli and absence of staining in the glomeruli (A-C: 660x), D. Positive staining near glomeruli at lower magnification (270X). E. Autoradiographic image of rat kidney sections (24 hr after 1251-streptavidin injection) showing strong peripheral radioactivity (Xl). 1332 Schechter et al: Streptavidin acci4mulation in the kidney

100 Immunohistochemistiy Immunoperoxidase staining of streptavidin in mouse renal 80 sections was carried out 24 hours following streptavidin injection (Fig. 6). Staining with anti-streptavidin (polyclonal) antibody and peroxidase-labeled goat anti-rabbit antibody revealed sequestra- 60 tion of streptavidin in proximal tubules mainly near the capsule. Positively stained tubules could be found also around the glomer- 40 uli whereas the absence of staining in the glomeruli was evident. Autoradiographic image of rat kidney sections (24 hr after 1251-streptavidin injection) showed strong peripheral radioactivity. 20 Use of streptavidin for targeting to the kidney > 0 Preliminary experiments to estimate the ability of streptavidin to target therapeutic doses of radionuclides were performed in 100 order to verify the anticipated high kidney/other tissue ratio. These experiments were performed in athymic nude mice (which 80 will serve in the future as carriers of human-derived tumors in pre-clinical experiments). Two streptavidin preparations radio- iodinated with radioisotopes of different decaying dose rates, that 60 is, 125! or 131! (half-time 60 and 8 days, respectively, 0.1 mCi! mouse), were administered i.p. into two groups of mice. 1251-BSA 40 was used as control. External radioactivity was monitored during a period of several days at the kidney or head regions of the intact mice. As is illustrated in Figure 7 top, 125j radioactivity persisted 20 in the kidney region for three days and declined gradually to 43% on day 4 and to 37% on day 5. For comparison, radioactivity at the head region declined to 17% of the original level already after one 0 day. Kidney radioactivity of 1251-BSA was low. 1311-streptavidin 0 2 4 6 8 (Fig. 7 bottom) persisted for a shorter period of time and was Time,days after injection down to 45% on day 2 and to 11% on day 4. Fig. 7. Monitoring external radioactivity in athjmic nude mice following A preliminary study was designed to demonstrate the targeting '251-streptavidin or 1311-streptavidin injection. Athyiiic nude mice (7 mice! of biotinylated ligands via streptavidin. Biotinyl-tyrosine (b-Y), group) were injected i.p. with 0.1 mCi of radioactive streptavidin and biotinyl-alanyl-alanyl-tyrosine (b-AAY) and b-a-MSH (11 resi- monitoring was performed at the kidney (•)andhead (0) regions.dues long peptide), radio-iodinated at their tyrosine residues, '251-labeled BSA was used as control (A). Data are expressed as mean SD. were allowed to interact with cold streptavidin. The resulting radioactivity units complexes were separated from free iodide or uncomplexed 1251-biotinyl-ligand by size exclusion chromatography. As shown in Figure 8, radioactive b-Y and b-AAY preferentially accumu- lated in the kidney, their biodistribution and time dependent accumulation in the kidney, 300.tg caused a reduction of 35% inaccumulation (Fig. 9) being comparable to that of 1251-streptavi- the radioactivity accumulated per gram of kidney tissue. Sincedin itself. Accumulation of b-a-MSH was short-lived, with 40%/g 20% of the injected dose was retained in each kidney, it can beat seven hours post-injection which declined to 15%/g at 24 and 48 calculated that at 24 hours after injecting 300 g streptavidin 40hours (Figs. 8 and 9). ig of the protein were retained in each kidney. Discussion Streptavidin distribution in kidney medulla and cortex The present study describes the specific accumulation of To examine, whether streptavidin accumulation in the kidney isstreptavidin in the kidney and demonstrates its potential use as a confined to restricted regions, mice were injected with 1251 targeting vehicle to the kidney for specific renal delivery of streptavidin and at set time intervals their kidneys were removed,therapeutic agents. Streptavidin accumulates preferentially in the counted for total radioactivity and dissected under a microscopekidney cortex and is retained there for a period of three to four into the cortex and medulla sections (Fig. 5). Values in terms ofdays. Uptake values recorded in different experiments with several %/gtissuefor total kidney, medulla and cortbx were 76, 43 and 92mouse strains as well as with two other animal species (rat and at 24 hours and 57, 37 and 97 at 48 hours, respectively, indicatingrabbit) indicated that approximately 15% of the total injected preferential accumulation in the cortex. A more accurate evalua-dose can be accumulated in each kidney, an organ which com- tion of cortex and medulla distribution could be obtained in theprises only 1% of total body weight. In terms of protein capacity, rabbit (at 24 hr), with values for kidney, medulla and cortex of 2.3,40 xg of the protein can be retained in a mouse kidney, a value 0.2 and 3.14 %Ig tissue, respectively (1.4%/g for intermediarywhich is even higher in terms of tissue specificity since streptavidin tissue). accumulation is confined to the kidney cortex. The high and Schechter et al: Streptavidin accumulation in the kidney 1333

80

60 Cl) U) 0) 40

.E 20 Fig.8. Taigeting (biodistribution) of '251-b-Y, '25Jby and 1251-b-n-MSH to the kidney following their complexing to non-radioactive streptavidin. Uncomplexed ligands were down to 0 1 to 2% at 2 hours following injection. Data for pairs of mice are in Figure 1. Symbols are: (U) b-Y-St b-AAY-St b-MSH-St kidney; ()spleen;(EEl) liver; ()heart;(El) 24hr 24hr 7hr lung; (11) blood.

A B 100 100

80 80 =U) U) 60

40 - 40 160 20 - 20

Fig. 9. Time dependent retention in the kidney of 0 (A) 1251-streptavidin (U), 1251-b-Y (VA) and (B) 17 24 40 60 88 '25JbaMSH complexed to non-radioactive 7 24 48 96 streptavidin. Data for pairs of mice are in Time, hours Figure 1.

prolongedkidney retention of streptavidin in comparison to theAlso, the observation that native streptavidin as well as avidin, low levels in blood and other organs provides exceptionally highboth displaying biotin-binding properties similar to that of strepta- "target" to "non-target" ratios. Streptavidin may thus serve as avidin, did not bind to the kidney, supports the assumption that drug carrier for site-specific delivery or release of radionuclides,biotin or biotinyl groups do not serve as the major anchors for anti-viral or anti-inflammatory drugs, antibiotics, diuretics, che-streptavidin binding in the kidney. Thus, a major advantage of moterapeutic agents and other effector molecules. Specific target-streptavidin is that in addition to its high kidney accumulation, it ing to the kidney may increase drug levels in this organ whileis also equipped with extremely efficient built-in binding sites for decreasing general toxicity and adverse side effects. Drug attachedbiotin which are not required for its retention in the kidney. A to streptavidin via appropriate carrier molecules can be pro-large variety of biotinylated ligands such as proteins (antibodies, grammed for gradual and slow release as has been shown forenzymes, cytokines), peptides, drugs, toxins, radionuclides, fluo- other macro-molecularized drug systems [16—21]. rescent tags, etc. can be targeted to the kidney via streptavidin. Streptavidin retention in the kidney could he due to interactionThe biotin-ligand conjugate may be designed to permit sustained with endogenous biotin (10 nM in mice and 2 n in human) [221,release of the ligand by inclusion of appropriate cleavable or orbiotinyl-enzymes [231thatmight have been enriched in thedissociable chemical moieties between the biotin and the reactive kidney. However, addition of biotin to streptavidin prior togroup, or else to ascertain a stable bond [4]. injection did not significantly affect its biodistribution pattern. The high kidney uptake of streptavidin could be attributed to 1334 Schechteret al: Streptavidin accumulation in the kidney

the resistance property of streptavidin, whether or not Acknowledgments interaction with specific sites is involved. Recent studies describe cell-adsorptive properties of the streptavidin molecule indepen- This research was supported by grants from the Israeli Academy of dent of biotin [24—26]. The binding, which is low in the native Science and Humanities and the Baxter Foundation. form but is enhanced by truncation and biotin complexing, appears to be linked to an RYD-containing sequence in strepta- vidin. This sequence is highly homologous to the RGD domain of Reprint requests to B. Schechter, Department of Chemical Immunology, fibronectin and other adhesion proteins which interact with cell The Weizmann Institute of Science, Rehovot, Israel 76100. surface integrins [27—29]. Following interaction with endogenous biotin, streptavidin may reach the kidney and interact with cell-surface adhesion receptors via RGD mimicry. References Another mechanism could be related to the processing of low molecular weight proteins (lower than 64 kDa) which are filtered 1. CHAlET L, WOLF FJ: The properties of streptavidin, a biotin-binding through the glomeruli and undergo a process of tubular endocy- protein produced by streptomyces. Arch Biochem Biophys 106:1—5, tosis and active lysosomal degradation [301.Theseproteins first 1964 interact with specific sites of the luminal membrane and are 2. GREENNM:Avidin, in Advances in Protein Chemistiy, edited by internalized by endocytic vacuoles. Proteolytic enzymes and acidic ANFINSENCB,EDSAL JT, RICHARD FM, New York, Academic Press, 1975, p 85 pH accelerate their catabolism in the lysosomes, whereas mem- 3. WILCHEK M, BAYER EA: The avidin-biotin complex in immunology. brane transport systems are involved in the active secretion and Immunol Today 5:39—43, 1984 reabsorption of degradation products such as amino acids. 4. WILCHEK M, BAYER E: The avidin-biotin complex in bioanalytical Streptavidin may belong to this category of proteins since immu- applications (review). Anal Biochem 171:1—32, 1988 5. HNATOWICH D, VIRZI F, RUSCKOWSKI M: Investigations of avidin and nohistochemical staining of streptavidin-containing kidneys with biotin for imaging applications. J NucI Med 28:1294—1302, 1987 anti-streptavidin antibody indicated that streptavidin indeed ac- 6. PAGANELLI G, RIvA P, DELEIDE G: In vivo labeling of biotinylated cumulates in proximal tubular cells. Entrapment of streptavidin in monoclonal antibody by radioactive avidin: a strategy to increase tubular cells following glomerular filtration may be due to its tumor radiolocalization. mt j Cancer Suppl 2:121—125, 1988 enzyme resistance and delay in degradation. This protein, though 7. OEHR P, WESTERMANN J, BIERSACK HJ: Streptavidin and biotin as falling within the upper limit of the molecular size range, may potential tumor imaging agents. J Nuci Med 29:728—729, 1988 8. SINISTSYN VV, MAMONTOVA AG, CHECKNEVA YY, SHNYRA AA, benefit from its tightly rigid structure which becomes even more DAMOGATSKY SP: Rapid blood clearance of biotinylated IgG after condensed upon interaction with endogenous biotin. infusion of avidin. J Nuci Med 30:66—69, 1989 It has been suggested that such low molecular weight proteins 9. SCHECHTER B, A1NONR,WILCHEK M, SCHLESSINGER J, HURWITZ E, may serve as efficient drug carriers that release drugs site- ABOUD-PIRAK E, SEi... M: Indirect immunotargeting of cis-Pt to human epidermoid carcinoma KB using the avidin-biotin system. mt J specifically in the kidneys upon catabolism [31]. Although this Cancer 48:167—172, 1991 type of drug targeting may be beneficial in certain cases, it is still10. HASHIMOTO N, TAKATSU K, MASUHO Y, KISHIDA K, HARA T, a short-term transient event manifested via a relatively rapid HAMAOKA T: Selective elimination of a B cell subset having acceptor uptake and degradation of the drug protein. The use of an site(s) for T cell-replacing factor (TRF) with biotinylated antibody to enzyme-resistant protein such as streptavidin, irrespective of its the acceptor site(s) and avidin-ricin A-chain conjugate. J Immunol renal accumulation mechanism, may offer certain advantages such 32:129—135, 1984 11. ARGARANA CE, KUNTz ID, BIRKEN S, AXEL R, CANTOR CR: Molec- as longer maintenance of therapeutic or diagnostic ligands as well ular cloning and nucleotide sequence of the streptavidin gene. Nucl as improved local kinetics of endorenally released drugs. Co- Acid Res 14:182—187, 1986 valently bound radionuclides, which persists in the kidney for 12. SCHECHTER B, SILBERMAN R, ARNONR,WILCFIEK M: Tissue distri- three to four days, may be utilized for streptavidin-mediated bution of avidin and streptavidin injected to mice. Eur J Biochem radiotherapy. Biotinylated ligands, on the other hand, may be 462:327—331, 1990 13. BAYER EA, BEN-HUR H, HILLER Y, WILCHEK M: Postsecretoiy used for controlling drug releasability dependent on the length of modifications of streptavidin. Biochem J 259:369—376, 1989 the spacer: a small directly bound ligand such as b-Y (where14. WILCHEK M, BAYER EA: Avidin-biotin technology ten years on: has it tyrosine is radio-iodinated) accumulates exclusively in the kidney lived up to its expectations? Trend Biochem Sci 14:408—412, 1989 and is gradually released due to eventual degradation of the 15. HUNTER WM, GREENWOOD FC: Preparation of iodine-131 labeled streptavidin-carrier. Even tyrosine spaced by two alanyl residues growth hormone of high specific activity. 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SCHECHTER B, PAUZNER R, WILCHEK M, ARNON R: Cis-platinum(II) intermediary linkers may enable tailor-made attachments of ii- complexes of carboxymethyl-dextran as potential antitumor agents. II. gands to streptavidin to provide high and prolonged organ In vitro and in vivo activity. Cancer Biochem Biophys 8:289—298, 1986 maintenance, or else enhanced release. 19. SCHECHTER B, WILcHEK M, ARNONR:Cis-platinum(II) complexes of Schechter et al: Streptavidin accumulation in the kidney 1335

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