Streptavidin in Antibody Pretargeting. 4. Site ... - ACS Publications

Bioconjugate Chem. 2004, 15, 1454−1463

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Streptavidin in Antibody Pretargeting. 4. Site-Directed Mutation Provides Evidence That Both Arginine and Lysine Residues Are Involved in Kidney Localization D. Scott Wilbur,*,† Donald K. Hamlin,† James Sanderson,† and Yukang Lin§ Department of Radiation Oncology, University of Washington, Seattle, Washington 98195, and Fred Hutchinson Cancer Research Center, Seattle Washington 98109. Received June 5, 2004; Revised Manuscript Received October 4, 2004

The in vivo application of the protein streptavidin is limited by its propensity to localize to kidney, particularly when it is used as a carrier of radionuclides in Targeted Radionuclide Therapy. Our previous studies demonstrated that modification of recombinant “core” streptavidin (rSAv) by reaction of lysine residues with succinic anhydride and arginine residues with 1,2-cyclohexanedione dramatically decreases the kidney concentrations over that obtained with wild-type rSAv. In this investigation, we explored the role of lysine and arginine residues in kidney localization further by evaluating sitedirected mutants of rSAv. In the five mutants studied, the four lysine residues found in each subunit of rSAv were replaced (independently) with an alanine (K80A, K121A, K132A, K134A), and a specific arginine was replaced with a histidine (R59H). The rSAv mutants were prepared from a “core” rSAv construct produced by expression in E. coli that had 124 amino acids (residues 13-136). Another rSAv construct that had 127 amino acids (residues 13-139), used in most of our previous studies, was also included for comparison. As an additional comparison, succinylated rSAv was prepared and evaluated. The rSAv proteins were radioiodinated and injected into athymic mice that were on a biotinfree diet for 5-7 days prior, and biodistribution data were obtained (for most proteins) at 1, 4, 24, and 48 h postinjection. The data obtained show large differences in kidney localizations of the wildtype rSAv and some rSAv mutants. The largest difference in the kidney concentration was noted for the rSAv-K134A mutant (1.90 ( 0.22%ID/g; 24 h pi) as compared to the wild-type rSAv (31.83 ( 5.26%ID/g) at the same time point. The concentration of rSAv-K134A mutant in kidney was slightly lower than that obtained with succinylated rSAv. At the 24 h time point, the kidney concentrations of the rSAv-R59H mutant (8.95 ( 2.94%ID/g) and the rSAv-K121A mutant (11.86 ( 1.61%ID/g) were lower than wild-type rSAv, but the rSAv mutants rSAv-K80A (27.95 ( 1.82%ID/g) and rSAv-K132A (32.50 ( 10.09%ID/g) were essentially the same. The data suggests that specific lysine and arginine residues are involved in kidney localization. Possible mechanisms for the observed kidney localization are discussed.

INTRODUCTION

(1013-1015

M-1)

The very high binding affinity of the egg white protein avidin (Av)1 (2) and the bacterial protein streptavidin (SAv) (3) with biotin make them valuable chemical tools for a wide variety of applications. While most of the applications developed thus far are in vitro assays (4-10), in vivo applications are also being developed. For in vivo applications, protease resistant “core” recombinant SAv (rSAv) is being used (11). An inherent problem for use of the proteins Av and rSAv in vivo is their propensity to localize to kidney and liver. * Address correspondence to D. Scott Wilbur, Ph.D., Department of Radiation Oncology, University of Washington, Box 359658, 325 Ninth Ave., Seattle, WA 98104-2499. Phone: 206341-5437. Fax: 206-341-5438. E-mail: [email protected]. † University of Washington. § Fred Hutchinson Cancer Research Center. 1 Abbreviations: Av, avidin; biotin-Rhd, biotin derivative containing a rhodamine moiety; biotin-CNCbl, biotin derivative containing a cyanocobalamin moiety; ChT, chloramine-T; cpm, counts per minute; IEF, isoelectric focusing; mAb, monoclonal antibody; %ID/g, percent injected dose per gram; PBS, phosphatebuffered saline; pi, postinjection; rSAv, recombinant streptavidin; rt, room temperature, Sav, streptavidin; sSAv, succinylated recombinant streptavidin.

Importantly, there is a marked difference in the in vivo characteristics of the two proteins. Av clears rapidly from blood and is localized in both kidney and liver, whereas rSAv clears from blood more slowly and localizes primarily in kidney (12, 13). The difference in pharmacokinetics and tissue localization of these similar proteins comes from Av being glycosylated and having a much higher pI than SAv (i.e. pI ∼10 vs 6-7). Deglycosylation of Av makes it more homogeneous (3) and results in blood clearance that is similar to rSAv, but the high pI can cause nonspecific interaction with tissues. Therefore, SAv is often considered more attractive for in vivo applications. The natural propensity for SAv to localize to kidney is problematic for its use in vivo, except perhaps in some unique applications (14). We are investigating the use of SAv in an approach to cancer therapy termed “pretargeting” (15-18). In those studies, SAv is radiolabeled and injected into mice bearing human prostate tumor xenografts, which have been pretargeted with a biotinylated antibody or antibody fragment (19). While we demonstrated that an equal molar quantity of radiolabeled rSAv can bind with the biotinylated antibody on the tumor, a majority of the radiolabeled rSAv localizes to kidney. In this situation, the high concentration of

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Kidney Localization of Streptavidin Mutants

radioactivity in the kidney precludes administration of an adequate quantity of a radionuclide to kill the tumor due a dose-limiting toxicity in the kidney. For this reason, we are investigating methods for alleviating the kidney localization of rSAv. In prior studies, we demonstrated that chemical modification of lysine amines by reaction with succinic anhydride (20) and modification of arginine guanidine groups by reaction with 1,2-cyclohexanedione (21) on rSAv resulted in decreased kidney concentrations with minimal affect on biotin binding. An alternative method for modifying lysine and arginine residues in rSAv is to conduct site-directed mutations in which lysine and arginine residues are replaced with other amino acids. Indeed a large number of sitedirected mutations of rSAv have been conducted with truncated or “core” SAv (11, 22) to probe interactions in the biotin-binding pocket (23-28) and produce mutants with desirable properties for specific applications (2936). As rSAv is made up of four identical subunits (3), site-directed mutation of one amino acid results in changing four amino acids in the tetrameric intact molecule. Importantly, decreased kidney concentrations were observed in previous studies involving high specific activity (i.e. very low concentration) acylation of lysine residues with the reagent, p-[125I]iodobenzoate N-hydroxysuccinimide ester, suggesting that modification of only one lysine residue per subunit might be required to decrease the kidney localization (13). It was reasoned that site-directed mutations which replace only one of the four lysines at a time could provide information as to whether this observation was correct, and if it were, identify which residue was responsible for kidney localization. Additionally, it had been postulated that kidney localization may be due to the 59RYD61 sequence in SAv (37). As previously mentioned, modification of arginine residues with 1,2cyclohexanedione demonstrated that kidney localization was dramatically decreased (21). Thus, site-directed mutation of that specific arginine2 (R59) might also provide confirmation that arginine was involved in kidney localization of rSAv. On the basis of these considerations, we have conducted an investigation in which the four lysine residues in each streptavidin subunit (K80, K121, K132, K134) were replaced with alanine residues, and the arginine residue of interest (R59) was replaced with a histidine residue. Following production of the rSAv mutants, they were radioiodinated and injected into athymic mice, and their concentrations in tissues (including kidney) were assessed at selected times (e.g. 1, 4, 24, and 48 h) postinjection (pi). The experimental methods used, and results obtained, in that investigation are described herein. EXPERIMENTAL PROCEDURES

General. All chemicals purchased from commercial sources were analytical grade or better and were used without further purification. Chloramine-T (ChT) and phosphate-buffered saline (PBS) were obtained from Sigma (St. Louis, MO). Recombinant streptavidin was obtained from Boehringer Mannheim (Indianapolis, IN). Iminobiotin affinity matrix was obtained from Pierce Chemical (Rockford, IL). Solvents for HPLC analyses were obtained as HPLC grade and were filtered (0.2 µm) prior to use. The biotin derivative containing the cyanocobalamin chromophore (biotin-CNCbl) was obtained3 as previously described (38). Rhodamine-derivatized biotin (biotin-Rhd) was prepared as previously described (39). Biotin deficient diet chow with 10% egg white solids (TD 96343) was obtained from Harlan Teklad (Madison, WI,

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www.harlan.com). Centricon-10 and -30 centrifugation concentrators were obtained from Amicon (Beverly, MA). PD-10 and NAP-10 (Sephadex G-25) size-exclusion columns were obtained from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Na[125I]I and Na[131I]I were purchased from PerkinElmer Life and Analytical Sciences (formerly NEN/ Dupont, Billerica, MA) as high concentration/high specific activity radioiodide in 0.1 N NaOH. Measurement of 125I and 131I was accomplished on the Capintec CRC-15R. Tissue samples were counted in a Wallac 1480 Wizard gamma counter with the following window settings: 15-85 keV for I-125 and 260 to 430 keV when 125I and 131 I are counted together. The 125I counts are compensated for spillover counts from the 131I in the calculations. Chromatography. FPLC purifications of the rSAv’s were performed on a Pharmacia 2000 FPLC system fitted with a 280 nm detector. Expression level and purification recovery were determined using biotin-Rhd binding (39) in conjunction with size exclusion separation on a Zorbax GF-250 column (Dupont) run at 1.0 mL/min using a mobile phase of 20 mM sodium phosphate, pH 7, containing 15% DMSO, 1 mM sodium azide, and 500 mM sodium chloride. The effluent was monitored at 547 nm using a Varian Dynamax PDA-2 detector, and the peak area corresponding to the rSAv elution was determined using a Varian Dynamax HPLC Data System (Walnut Creek, CA). HPLC separations of the modified rSAv were conducted on a Hewlett-Packard isocratic system consisting of a Model 1050 pumping system and a Model 1050 Multiple Wavelength Detector (280 nm). Proteins were evaluated on a Protein Pak 300 SW glass size-exclusion column (10 µm, 8 mm × 30 cm, Waters Corporation, Milford, MA) run at 1.0 mL/min eluting with 50 mM sodium phosphate buffer (pH 6.8) which contained 300 mM NaCl, 1 mM EDTA, and 1 mM sodium azide. Analyses of the HPLC data were conducted on HewlettPackard HPLC ChemStation software. Radiolabeled Proteins. Size-exclusion chromatography was also used to evaluate radioiodinated rSAv. HPLC separations of radioiodinated proteins were conducted on a system that consisting of a Model 1050 pumping system, a Waters Lambda-Max 481 UV detector, and a Beckman model 170 radioisotope detector. The detectors were connected to a computer through a Hewlett-Packard Model 35900 interface, and data from the chromatograms was obtained on a computer running Hewlett-Packard ChemStation Software. The same size-exclusion column and elution conditions were used for the radiolabeled proteins as unlabeled proteins. Mass Spectral Analyses. Electrospray mass spectra were obtained by atmospheric pressure chemical ionization on a Micromass Quattro II Tandem Quadrupole Mass Spectrometer (Micromass Ltd., Manchester, U.K.). The sample was introduced by direct injection. The mass spectral data was obtained on a PC running Windows NT based Micromass MaxEnt software. Prior to the mass spectral analyses, rSAv proteins were desalted. In the desalting procedure, 100 µL of a SAv in PBS solution was placed on a NAP-10 column that was equilibrated with deionized water. The column was eluted with deionized water, and the protein fractions were combined. The combined fractions were placed in a Centricon-30 ultrafiltration apparatus, concentrated, and washed three times with 1 mL of deionized water. The filtered solution was then concentrated to yield approximately 1 mg/mL of protein.

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Preparation of Streptavidin Mutants. Preparation of wild-type rSAv, rSAv mutants2 R59H, K80A, K121A, K132A, and K134C was accomplished in four steps: (1) construction of the rSAv mutant gene, (2) expression of rSAv mutant in E. coli, (3) refolding of rSAv proteins, and (4) purification. The procedures used in each step follow. (1) The site-directed mutagenesis was performed using the QuickChange kit (Stratagen, La Jolla, CA). Plasmid A140-9, which contains the wild-type of core SAv gene in a pET3a vector (Novagen, Madison, WI) was the starting construct. The reactions were carried out in 50 µL of supplied reaction mixture containing 400 nM of each oligonucleotide primer and 100 ng of plasmid DNA. Reactions were performed on a PTC-100 thermal cycler (MJ Reaction, Waltham, MA) with 1 cycle of 95 °C for 30 s and 17 cycles of the following profile: 95 °C for 30 s; 55 °C for 1 s; 68 °C for 12 min. The reaction mixture (3 µL) was used to transform E. coli XL-1 Blue competent cells (60 µL, Statagene). Plasmid DNA were prepared from cultures of the transformants using a QIAprep Spin Miniprep kit (Qiagen, Valencia, CA). The sequences were confirmed using a Big Dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). (2) Cultures of the plasmid-containing E. coli BL21 (DE3) pLysS (Novagen) were grown in shake flasks overnight at 37 °C in Terrific broth (40 mL; Invitrogen, Carlsbad, CA) containing 50 µg/mL of carbenicillin (Sigma Chemical, St. Louis, MO). The cultures were diluted ∼100-fold into fresh media (8 × 500 mL) contained in 2 L shake flasks and were grown until the cultures reached an optical density of 0.3-0.6 at 600 nm. At that point, isopropyl β-D-thiogalactopyranoside (Amersham Pharmacia Biotech Inc., Piscataway, NJ) was added to a final concentration of 0.5 mM. Incubation was continued for an additional 16 h. Cells were harvested by centrifugation at 4000g for 10 min at 10 °C and then washed twice with PBS (7 mM Na2HPO4, 150 mM NaCl, pH 7.0) before processing. (3) Cell pellets were resuspended in ice-cold TE (30 mM Tris, 1 mM EDTA at pH 8.0) at 25% w/v. The cells were disrupted through two cycles of microfluidization (M-110S, Microfluidics International, Newton, MA). The inclusion bodies were recovered by centrifuging the suspension at 15 000g for 90 min at 10 °C, and then the supernatant was discarded. Recovery of the mutants from the pelleted inclusion bodies was performed as described in the literature (35). Briefly, a microfluidized pellet was resuspended in 8 mL of ice-cold 8 M guanidine-HCl at pH 1.5 per liter of cell culture. The resuspension was dialyzed at 5-10 °C against 6 M guanidine-HCl at pH 1.5 to remove endogenous biotin. The dialysate was centrifuged at 10 000g for 20 min, and then the supernatant was added dropwise to vigorously stirred, ice-cold refolding buffer (4 mM KH2PO4, 16 mM Na2HPO4, 115 mM NaCl at pH 7.0) using a volume of at least 31× that of the supernatant volume. The suspension was incu2 Single letter designations for amino acids are used for simplification. Some single letter designations used in this paper include the following: R, arginine; K, lysine; A, alanine; H, histidine; G, glycine; D, aspartic acid; Y, tyrosine. Single letter designations for amino acids can be found in biochemistry text books (1). Site-directed mutations are designated by the one letter code for the amino acid that was originally present, followed by the residue number of that amino acid and then the single letter designation for the replacement amino acid. For example the abbreviation R59H designates that the arginine residue at amino acid 59 was replaced with a histidine residue.

Wilbur et al.

bated at 4 °C overnight without stirring, and spun at 7000g for 5 min at 10 °C. The pellet was discarded. (4) Prior to loading on the iminobiotin affinity column, the supernatant (from above) was adjusted to 50 mM glycine, 500 mM NaCl at pH 9.6, using concentrated stock. Conductivity was adjusted to 46-48 mSiemens/ cm with deionized water, and the solution was filtered through a 2 µm membrane overlaid with a thin film of Cell Pure P-100 diatomaceous earth. The iminobiotin matrix was packed in a column and was equilibrated in 50 mM glycine, 500 mM NaCl at pH 9.6. The capacity for rSAv was >2 mg/mL bed volume under a flow of 3 mL/cm2/min. The refolded rSAv mutant was loaded at room temperature onto a column which contained 150 mL of matrix bed volume per 75 g of cells. After washing with 20-bed volumes of 50 mM glycine, 500 mM NaCl at pH 9.6, the rSAv was eluted using 0.2 M sodium acetate, 0.1 M NaCl at pH 4.0. The eluate was neutralized to pH 7 with 300 mM Tris buffer (pH 8.0) and then exhaustively dialyzed in PBS at 4 °C. It was subsequently sterile filtered for refrigerated storage at 4 °C. Samples from the refolding and purification steps were run on 4-20% SDS-PAGE gels (Invitrogen, Carlsbad, CA) to qualitatively assess the rSAv expression levels and recovery of the tetrameric rSAv mutants. Quantitative assessment was performed using biotin-Rhd by size-exclusion chromatography. The final concentration of rSAv was calculated by comparison with a rSAv standard analyzed under the same conditions. Succinylation of Streptavidin. The succinylation reactions were conducted in a manner similar to that previously described (20). Briefly, a 200 µL aliquot of a 5 mg/mL solution of rSAv (1 mg, 1.9 × 10-2 µmol) was added to 370 µL of 50 mM NaHCO3 buffer (pH 8.5). To this solution was added either 50 equiv (94 µg; 0.94 µmol) or 100 equiv (188 µg; 1.88 µmol) of succinic anhydride in 20 µL DMSO. After 30 min at room temperature, the contents were transferred to a Centricon-10 and concentrated to approximately 50 µL. PBS (500 µL) was added and the solution was again concentrated to 50 µL. This wash step was repeated five times. The final concentrations of protein (e.g. 3.2 mg/mL) and recovery yields (8085%) were determined for each succinylation reaction. Isoelectric Focusing. IEF were obtained on a Novex PowerEase 500 instrument with the XCell II chamber using Invitrogen (Novex) precast gels, pH 3-10 or pH 3-7 (1.0 mm × 12 well) running under the standard IEF Program. The protein was stained with GelCode Blue Stain (Pierce). IEF standards were from Serva Electrophoresis GmbH: pI ) 10.7 - cytochrome C; 9.5 ribonuclease A; 8.3, 8.0, 7.8 - lectin; 7.4, 6.9 - myoglobin; 6.0 - carbonic anhydrase; 5.3, 5.2 - β-lactoglobulin; 4.5 - trypsin inhibitor; 4.2 - glucose oxidase; 3.5 - amyloglucosidase. Biotin-Binding Studies. Biotin binding studies were conducted using biotin-CNCbl3 as previously described (38). Briefly, to 300 µL of a 1 mg/mL solution (300 µg; 5.65 nmol) of wild-type rSAv or a rSAv mutant in a plastic microcentrifuge vial was added a freshly prepared solution containing approximately 4 equiv (22.6 nmol) of biotin-CNCbl dissolved in 30 µL of 10% aqueous DMSO. The rSAv/biotin derivative mixture was incubated for 1 h at room temperature and 100 µL of the solution was assayed on the HPLC. To the remaining rSAv/biotin derivative solution was added 40 µL of a 1 mg/mL 3 The biotin-CNCbl reagent is commercially available from Quanta BioDesign (Powell, OH, www.quantabiodesign.com) as biotin-dPEG4-cyanocobalamin.

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Kidney Localization of Streptavidin Mutants

Figure 1. Amino acid sequence of the “core” streptavidin used. Wild-type rSAv (residues 13-136) has the sequence shown, with the exception that residue number 13, which is an alanine in native SAv, was replaced with a methionine to initiate translation. The amino acid residues where site-directed mutations were performed (R59H, K80A, K121A, K132A, K134A) are circled in the sequence.

solution (40 µg; 164 nmol) of biotin in 10% aqueous DMSO. That solution was incubated for 4 h and 100 µL was removed for analysis by HPLC. Areas under the peaks for bound and unbound biotin derivative were evaluated to determine if a change in the percent bound had occurred. Due to difficulties in assessing the quantity of rSAv mutant present, in some examples there was a slight excess (30 Å). The pairing effect of arginine and lysine residues is due to the symmetrical nature of the tetrameric rSAv molecule. Graphical depictions of the proximity of lysines 121, 134, and arginine 59 to one another are provided as Figures S17 and S18 in the Supporting Information. Molecular modeling using space filling views also show the positive charges of these residues to be surface exposed. While it is apparent from the results of these studies that arginine 59 and lysine 121 have an influence on the kidney localization of rSAv, we have not provided evidence that the nature of their location, and/or distance between residues of two positive charges, is important in the renal cell endocytosis process. Further studies will be required to answer such questions. Summary. It is apparent from this investigation that some lysine residues affect the kidney localization of rSAv more than others. In particular, the most accessible lysine (K134) near the carboxyl terminus of each subunit has the largest effect on kidney localization when it is replaced with alanine. This may indicate that it is essential in the initial recognition/binding in kidney. Another lysine residue (K121) has an effect on kidney localization, but it is much less. This may indicate that it is also involved in the binding/internalization process, but it only occurs if the terminal lysine (K134) binds first. The arginine residue studied (R59) also has an effect on the kidney localization that appears to be secondary to the binding of the terminal lysine (K134), although the overall effect of its replacement with a histidine is comparable to that observed with succinylated rSAv. No data was obtained for other arginine residues, but these may also contribute to the kidney localization. These data do not provide a proof of the mechanism of kidney localization, but the location of the arginine and lysine residues within the rSAv structure appears to have a significance effect regarding kidney localization. Most importantly, we have found that mutation of only one lysine residue (per subunit) can provide a rSAv molecule that has a very low sequestration in the kidney. ACKNOWLEDGMENT

We thank Janna Quinn and Dr. Robert Vessella for their contributions to the biodistribution studies. We thank NeoRx Corporation for their contribution to these studies. The rSAv mutants were prepared at NeoRx Corporation when two authors (J.S., Y.L.) were employed in the company. We are grateful for the financial support provided by the Department of Energy, Medical Applications and Biophysical Research Division, Office of Health and Environmental Research under grant number DEFG03-98ER62572. Supporting Information Available: Complete biodistribution data for wild-type rSAv, succinylated rSAv, and rSAv mutants (Tables S1-S8); size-exclusion HPLC and mass spectra for rSAv, rSAv mutants and succinylated rSAv (Figures S1-S16); and graphical representations of the proximity of paired lysines 121/134 and arginine 5 The lysine amines in K134 have free movement so a close proximity can be attained; however, the groups are not held in that configuration. The location of the amines shown in Figure 17 (Supporting Information) are not established, as those amino acids were added to the crystal structure data for molecular modeling purposes.

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