Streptavidin in Antibody Pretargeting. 3. Comparison of Biotin Binding

Recombinant streptavidin (rSAv) is of interest as a carrier of α-emitting ... increase in the biotin derivative dissociation rate was noted for argin...
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Bioconjugate Chem. 2002, 13, 611−620

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Streptavidin in Antibody Pretargeting. 3. Comparison of Biotin Binding and Tissue Localization of 1,2-Cyclohexanedione and Succinic Anhydride Modified Recombinant Streptavidin D. Scott Wilbur,*,† Donald K. Hamlin,† Damon L. Meyer,|,⊥ Robert W. Mallett,| Janna Quinn,‡ Robert L. Vessella,‡ and Oliver W. Press§ Departments of Radiation Oncology and Urology, University of Washington, Seattle, Washington 98195, and Fred Hutchinson Cancer Research Center, Seattle Washington 98109, and NeoRx Corporation, Seattle, Washington 98119. Received November 1, 2001; Revised Manuscript Received February 1, 2002

Recombinant streptavidin (rSAv) is of interest as a carrier of R-emitting radionuclides in pretargeting protocols for cancer therapy. Due to the inherently high kidney localization of rSAv, modification of this protein is required before it can be useful in pretargeting. Previous studies (Wilbur, D. S., Hamlin, D. K. et al. (1998) Bioconjugate Chem. 9, 322-330) have shown that succinylation of rSAv using succinic anhydride decreases the kidney localization appreciably. In continuing studies, the biotin binding characteristics and biodistribution in mice of rSAv modified by reaction with succinic anhydride (amine modification) or 1,2-cyclohexanedione (arginine modification) have been compared. Modification of rSAv was conducted using 5-50 mol equiv of succinic anhydride and 60-200 mol equiv of 1,2-cyclohexanedione. Most studies were conducted using rSAv modified with the highest quantities of reagents. Succinylation of rSAv did not alter binding with biotin derivatives, but a small increase in the biotin derivative dissociation rate was noted for arginine-modified rSAv. Amino acid analysis of 1,2-cyclohexanedione-treated rSAv indicated about 40% of the arginine residues, or an average of 1.6 residues per subunit, were modified, whereas none of the lysine residues were modified. IEF analyses showed that the pI of the arginine-modified rSAv was 5.3-6, whereas the pI for the succinylated rSAv was approximately 4. Electrospray mass spectral analyses indicated that one to three conjugates of 1,2-cyclohexanedione, and two to three conjugates of succinic anhydride, were obtained per subunit. Both modification reactions resulted in greatly decreasing the kidney localization of rSAv (normally 20-25% ID/g at 4, 24, and 48 h pi). However, the kidney concentration for the succinylated rSAv continued to decrease (5% ID/g to 1.5% ID/g) from 4 to 48 h pi, whereas the concentration (5% ID/g) remained constant over that period of time for the arginine-modified rSAv. In contrast to this, the liver concentration appeared to be slightly higher (3% ID/g vs 2% ID/g) at the later time points for the succinylated rSAv. When less than 50 mol equiv of succinic anhydride were employed in the modification of rSAv, a correlation between increasing kidney localization with decreasing equivalents reacted was observed. Although the differences in the two modified rSAv are not substantial, succinylated rSAv appears to have more favorable properties for pretargeting studies.

INTRODUCTION

Selective targeting of therapeutic radionuclides to cancer cells in patients, termed “Targeted Radiotherapy” or “Endoradiotherapy” holds great potential for cancer therapy, particularly in the killing of disseminated cancer cells present in metastatic disease. Radiolabeled monoclonal antibodies (mAbs1) have been shown to be very * Address correspondence to D. Scott Wilbur, Ph.D., Department of Radiation Oncology, University of Washington, 2121 N. 35th Street, Seattle, WA 98103-9103, Phone: 206-685-3085, FAX: 206-685-9630, E-mail: [email protected]. † Department of Radiation Oncology, University of Washington. ‡ Department of Urology, University of Washington. § Fred Hutchinson Cancer Research Center. | NeoRx Corporation. ⊥ Current address for D. Meyer is Seattle Genetics, Inc. Bothell, WA. 1 Abreviations: ChT, chloramine-T; cpm, counts per minute; IEF, isoelectric focusing; mAb, monoclonal antibody; %ID/g, percent injected dose per gram; PBS, phosphate-buffered saline; pi, postinjection; rSAv, recombinant streptavidin; rt, room temperature, SAv, streptavidin.

effective at targeting cancer cells, but inherent problems have, thus far, limited their use in cancer therapy (16). To circumvent the problems associated with directly labeled mAbs, alternative approaches utilizing the cancer cell targeting of mAbs are under investigation. One particularly promising approach is termed “pretargeting” (7,8). In that approach, a mAb conjugate is used to target cancer cells and a separate molecule that binds with the mAb conjugate is used to carry the therapeutic radionuclide. Although this alternative approach involves multiple administrations of reagents, it permits optimization of important parameters, such as pharmacokinetics, route of excretion, and stabilization of the radionuclidecarrying molecule from metabolic processes. Indeed, promising therapy results have been obtained with the pretargeting approach (9-11). In our previous studies, a pretargeting approach which uses biotinylated mAb Fab′ fragments with radiolabeled recombinant streptavidin (rSAv) was investigated (12). Those studies demonstrated that each biotinylated Fab′ bound in the tumor xenograft captured one radiolabeled rSAv molecule. However, an inherently high kidney localization of SAv molecules (13) was problematic with

10.1021/bc015574n CCC: $22.00 © 2002 American Chemical Society Published on Web 04/23/2002

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the approach. It has been hypothesized that the kidney localization observed comes from specific recognition of a surface RYD (Arg, Tyr, Asp) amino acid sequence which is similar to the fibronectin binding RGD (Arg, Gly, Asp) sequence (14,15) and results in a specific localization in the renal cortex (16). Studies in this laboratory demonstrated that the kidney localization could be alleviated by succinylation of rSAv (17). This result is thought to due to the high anionic surface charge produced on rSAv and not to an alteration in the RYD sequence on the surface of the protein. Our continued interest in the use of rSAv as a carrier of radionuclides in pretargeting protocols led us to further investigate methods of decreasing kidney localization of this protein. We were particularly interested in the chemical modification of the RYD sequence in rSAv investigated by personnel at NeoRx Corporation.2 They found that specific chemical modification of arginine residues using 1,2-cyclohexanedione (18-20) decreased the kidney localization. Those results prompted us to conduct an investigation comparing the biotin binding properties and tissue distributions of rSAv modified using succinic anhydride and 1,2-cyclohexanedione. The results of that investigation are reported 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). 1,2-Cyclohexanedione, and most other chemicals, were obtained from Aldrich Chemical Co. (Milwaukee, WI). Recombinant streptavidin was obtained from Boehringer Mannheim (Indianapolis, IN). Solvents for HPLC analysis were obtained as HPLC grade and were filtered (0.2 µm) prior to use. The biotin derivatives 1-3, containing the cyanocobalamin chromophore, were obtained as previously described (21). Biotin-deficient diet chow with 10% egg white solids (TD 96343) was obtained from Harlan Teklad (Madison, WI, www.harlan.com). Centricon-10 centrifugation concentrators were obtained from Amicon (Beverly, MA). Na[125I]I and Na[131I]I were purchased from 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: 1585 keV for I-125 and 260-430 keV when 125I and 131I are counted together. The 125I counts are compensated for spillover counts from the 131I in the calculations. Chromatography. HPLC separations of the modified rSAv were obtained on 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 0.75 or 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. Analysis of the HPLC data was conducted on Hewlett-Packard HPLC ChemStation software. Retention times (tR) for rSAv, arginine-modified rSAv, and succinylated rSAv were 27.6-27.8 min (0.75 mL/min flow) or 15.4-15.6 min (1.0 mL/min flow). 2 Mallett, R., and Meyer, D. NeoRx Corporation, Seattle, WA, unpublished results.

Wilbur et al.

Radiolabeled Proteins. Size-exclusion chromatography was also used to evaluate radioiodinated rSAv. HPLC separations of radioiodinated proteins were conducted on a system that consisted of a Waters Model 510 pump, a Waters Lambda-Max 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 proteins were separated on a BioSelect SEC250-5 size exclusion column (7.8 mm × 300 mm, BioRad, Hercules, CA), eluting with a solvent mixture of 25 mM Tris-HCl, 75 mM NaCl, and 1 mM EDTA, pH 7.2, at a flow rate of 0.70 mL/min. Retention time (tR) for rSAv was 13.4 min on this system. 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 directly. The mass spectral data was obtained on a PC running Windows NT based Micromass MaxEnt software. Arginine Modification of Streptavidin. Two experimental procedures for modification of rSAv were used in the studies. All studies used modified rSAv produced by Method A, except for the amino acid analyses. Method A. To 0.8 mL of rSAv (24.2 mg/mL, 19.4 mg, 0.37 µmol) in PBS was added 0.2 mL 1.0 M NaOH. To this mixture was added 8.6 mg (77 µmol) of solid 1,2cyclohexanedione. The mixture was stirred for 1 h and then eluted through a Sephadex G-25 column using a 20 mM sodium phosphate, 60 mM NaCl, pH 7.0 solution. The protein gained a slight yellow coloration during the modification. Method B. To 0.4 mL of 25.4 mg/mL solution of rSAv (10.2 mg, 0.19 µmol) in PBS was added 8 µL of 10 M NaOH to prepare a final concentration of 0.2 M. To that solution was added 1.3 mg (11.4 µmol) 1,2-cyclohexanedione, and the reaction was allowed to proceed for 5 h at ambient temperature. The modified rSAv solution was exchanged into PBS using a G-25 size exclusion column. From this procedure, 8.3 mg (81%) of modified rSAv was obtained following the buffer exchange. Succinylation of Streptavidin. The succinylation reactions were conducted in a manner similar to that previously described (17). 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 varying quantities [5 equiv: 9.4 µg (9.4 × 10-2 µmol); 10 equiv: 19 µg (19 × 10-2 µmol); 25 equiv: 47 µg (4.7 × 10-2 µmol); 50 equiv: 94 µg (94 × 10-2 µmol)] of succinic anhydride in 2-20 µL DMSO. After 30 min at room temperature, the contents were transferred to a Centricon-10 and concentrated to 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 (2.4-3.6 mg/ mL) and recovery yields (92-100%) were determined for each succinylation reaction. Electrophoresis. Two electrophoresis instruments and methods were used in the studies. The IEF gel shown in Figure 1 was obtained on a Novex PowerEase 500 instrument with the XCell II chamber using Invitrogen (Novex) precast gels, pH 3-10 (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: pH 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,

Succinylated and Arginine-Modified Streptavidin

Bioconjugate Chem., Vol. 13, No. 3, 2002 613 Table 1. Relative Dissociation of Biotin Derivatives from rSAv and Modified rSAva

biotin derivative

rSAv, %

succinylated rSAv, %

argininemodified rSAv, %

biotin-CN-Cbl, 1 biotin-sarcosine-CN-Cbl, 2 biotin-aspartate-CN-Cbl, 3

96 9 91

99 58 91

85 8 80

a Values represent the percent of biotin derivative remaining bound after 3 h at room temperature in the presence of a large excess of biotin. Structures of biotin derivatives are shown in Figure 3.

Figure 1. Digitized image of IEF gel comparing pI of unmodified rSAv with succinylated and arginine-modified rSAv. Lanes 1 and 5: IEF standards (Serva IEF markers 3-10). Lane 2: unmodified rSAv (control). Lane 3: succinylated rSAv. Lane 4: arginine-modified rSAv.

5.2, β-lactoglobulin; 4.5, trypsin inhibitor; 4.2, glucose oxidase; 3.5, amyloglucosidase. The IEF gels shown in Figures 5A and 5B were obtained on a PhastSystem electrophoresis apparatus (Pharmacia LKB, Uppsala, Sweden) using the optimized method for IEF described in “Separation Technique File #100”. The IEF were run using PhastGel IEF 3-9 precast gels. Protein staining was accomplished by the silver stain method described in “Development Technique File #210”. IEF was conducted with samples running in opposite directions. The electrophoresis standards were from the Broad pI Kit (pI 3.5-9.3) (Amersham Pharmacia Biotech, NJ) and are composed of: pI 9.30, trypsinogen; 8.65, 8.45, 8.15, lentil lectin; 7.35, 6.85, myoglobin; 6.55, 5.85, carbonic anhydrase B; 5.20, lactoglobulin A; 4.55, trypsin inhibitor; 3.75, methyl red; 3.50, amylglucosidase. Amino Acid Analysis. Amino acid analyses of hydrolyzed 1,2-cyclohexanedione-modified and -unmodified rSAv were conducted according to the procedure described by Heinrikson R. L. et al. (22). The modified rSAv samples were hydrolyzed with 6 N HCl in the vapor phase at 200 °C for 75 min and then derivatized with phenyl isothiocyanate (PTC) using an Applied Biosystems Model 420 H automated hydrolyzer/derivatizer. The PTCAA mixture was next injected onto a reversed-phase C-18 column configured on an Applied Biosystems Model 130A HPLC system. The column effluent was monitored at 254 nm and the chromatogram analyzed using the Applied Biosystems Model 610 data acquisition system. Biotin Binding Studies. Biotin derivative binding studies were conducted as previously described (21). Briefly, to 300 µL of a 1 mg/mL solution (300 µg; 5.65 nmol) of rSAv, arginine-modified rSAv, or succinylated rSAv in a plastic microcentrifuge vial was added 4 equiv (22.6 nmol) of a biotin conjugate (1-3) dissolved in 30 µL of a 10% aqueous DMSO solution. The rSAv/biotin derivative mixture was incubated for 1 h at room tem-

perature, 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 solution (40 µg; 164 nmol) of biotin in 10% aqueous DMSO. That solution was incubated for 3 h, and 100 µL was removed for analysis by HPLC. Areas under the peaks for bound and unbound biotin derivative provided the relative dissociation values in Table 1. In some examples, there was a slight excess (12

equiv of succinic anhydride were radiolabeled with 125I (four preparations). The specific activities of 125I-labeled succinylated rSAv preparations were: 5 equiv, 0.74 µCi/ µg; 10 equiv, 0.79 µCi/µg; 25 equiv, 0.84 µCi/µg; 50 equiv, 0.93 µCi/µg. A control that contained unmodified radiolabeled [131I]rSAv was prepared for coinjection. The control 131I-labeled rSAv had a specific activity of 1.49 µCi/µg. Each mouse was injected with a mixture containing 15 µg succinylated [125I]rSAv and 15 µg [131I]rSAv. Tissue biodistributions were obtained at 24 h postinjection as described above. RESULTS

Modification of rSAv. Modification of rSAv was conducted by its reaction with either succinic anhydride in NaHCO3 buffer at pH 8.5 or with 1,2-cyclohexanedione in PBS containing 0.2 M NaOH, pH 12. Succinic anhydride can react with a number of protein functional groups (e.g., amines, thiols, phenols, and imidazoles). All of the succinic anhydride reactions with protein side chain functional groups are reversible in the basic media (19) except for the reaction of amine to form amide bonds, making this modification selective for amino groups on proteins. Reaction of proteins with 1,2-cyclohexanedione is highly selective for guanidine functionality of arginine. There are two reaction products that can be obtained, depending on the pH of the reaction solution. As depicted in Scheme 1, reaction of cyclohexanedione with arginine at pH 7-9 produces a bicyclic chemical adduct (adduct A). However, this reaction is not desirable for our application as the formation of that adduct is reversible, potentially resulting in some, or all, of the arginine residues being unmodified when tested in vivo. The syndiol, adduct A, may be stabilized by boric acid (18), but this reaction is also reversible making the stability in vivo still an issue. At high pH, a reaction occurs which results in the irreversible formation of a spiro-bicyclic compound (adduct B). Fortunately, rSAv is like avidin in that it is quite stable to highly alkaline conditions (25,26), making this reaction useful for our application. Characterization of Modified rSAv. The modified rSAv was analyzed by size-exclusion HPLC. Although there were small differences in retention times noted (e.g., 0.1-0.2 min), no reproducible link could be made for a modification resulting in a longer or shorter retention time. Because the modification reactions alter the charges on the protein, IEF analyses were conducted. An IEF comparing rSAv, succinylated rSAv and argininemodified rSAv is shown in Figure 1. The unmodified rSAv (lane 2) has two primary bands with pI of around 7.67.8. The succinylated rSAv (lane 3) has a primary band with a pI around 4.2 and two small bands with pI around 4.5. The arginine-modified rSAv (lane 4) has a primary band with a pI around 5.3 and a diffuse area between pI 5.3-6.0.

Figure 2. Digitized images of electrospray mass spectra of unaltered rSAv (2A), succinylated rSAv (2B), and argininemodified rSAv (2C). Note that in Figure 2B, unaltered rSAv has been added as an internal reference.

Electrospray mass spectral data was obtained on samples of rSAv, succinylated rSAv, and arginine-modified rSAv to obtain information on the number of conjugates per subunit. Mass spectra for these materials are shown as Figures 2A-C. The mass spectrum of unaltered wild-type rSAv (Figure 2A) shows the mass of a subunit is 13270.5 amu (total mass of tetramer is 53082 amu). The mass spectrum for succinylated rSAv is shown in Figure 2B. Note that rSAv was added as an internal standard (i.e., peaks at 13270 amu and 13294 amu). Aside from peaks for the rSAv standard, the major peaks for the modified rSAv subunits reacted with 50 equiv of

Succinylated and Arginine-Modified Streptavidin

Bioconjugate Chem., Vol. 13, No. 3, 2002 615

Figure 3. Structures of biotin derivatives 1-3 used in rSAv binding studies. The cyanocobalamin chromophore (360 nm) allows UV quantification without interference from protein absorbance (i.e., 280 nm). The biotin derivatives were obtained as previously described (21).

succinic anhydride have masses of 13471 amu (100%;+200 amu over rSAv) and 13571 amu (85%; +300 amu) with a number of additional peaks present being due to sodium adducts. Since conjugation of a succinic anhydride adds a mass of 100 amu to the protein, the mass spectral data indicates that the majority of subunits have two or three succinic anhydride molecules conjugated. The mass spectrum for arginine-modified rSAv is shown in Figure 2C. Conjugation of 1,2-cyclohexanedione to form adduct A (Scheme 1) increases the mass of the protein by 112 amu, but rearrangement to form adduct B (Scheme 1) results in a mass increase of only 94 amu. Reaction of 200 equiv of 1,2-cyclohexanedione with rSAv produces a mixture of modified proteins with mass peaks for modified subunits of 13366 amu (72%; +95 amu), 13383 (46%; +112 amu; B), 13459 (100%; + 188 amu), 13478 (53%; +207 amu), 13497 amu (48%; +226 amu); 13555 amu, (38%; +284 amu), 13569 amu (63%; + 299 amu) and 13569 (37%; +320 amu). The mass spectral data indicate that both adducts (A and B) are obtained under the conjugation conditions employed in this investigation. The data indicate that there were one to three adducts per rSAv subunit, and the molecular weight increases correlate with the two types of adducts being present on rSAv (from the listing above) in the following combinations; rSAv plus: B (+94 amu), A (+112 amu), BB (+188 amu), BA(+206 amu), AA(+224 amu), BBB(+282 amu), BBA(+300 amu), and BAA(+318 amu). The specificity for arginine versus lysine residues in the conjugation with 1,2-cyclohexanedione was assessed by conducting amino acid analyses. Unmodified rSAv was run as a control. The analyses indicated that about 40% of the arginine residues were modified (i.e., amount of arginine decreased) by reaction with 1,2-cyclohexanedione. This implies that an average of 1.6 arginine residues per subunit reacted. Importantly, no change in the quantity of lysine was found, confirming that the cyclohexanedione reaction was specific for arginine residues. The biotin binding of modified rSAv was of interest. Although it is important to diminish the kidney localization of rSAv for in vivo pretargeting application, it is most desirable that this is done with the retention of the very high biotin binding affinity. Three biotin derivatives containing the cyanocobalamin chromophore (compounds

1-3, Figure 3) were prepared as previously described (21), and their binding with rSAv, succinylated rSAv, and 1,2-cyclohexanedione modified rSAv was evaluated. All rSAv samples displayed similar association kinetics for the three biotin derivatives (data not shown). Of more interest were the relative rates of dissociation of rSAvbound biotin derivatives. In that assay, 4 equiv of the biotin derivative are bound with the rSAv, a large excess of biotin is added, and the amount of released (dissociated) biotin derivative is measured at a 3 h time point (27). The relative dissociation rates obtained in this investigation are listed in Table 1. The results indicate that succinylated rSAv retains a slow dissociation rate for the biotin-cyanocobalamin adduct 1, but argininemodified rSAv appears to have a slightly faster dissociation rate. In our previous studies, it was shown that biotin adducts containing a sarcosine (N-methylglycine) moiety (e.g., 2) have an increased dissociation rate. It was of interest to determine if the modified rSAv showed similar relative dissociation rates. Indeed, argininemodified rSAv displayed similar dissociation kinetics to that of unaltered rSAv. Interestingly, the succinylated rSAv appeared to have a slower dissociation rate. The R-aspartate adduct of biotin, 3, was also evaluated as biotin derivatives currently under investigation incorporate this structural feature to block the biotinamide cleaving action of biotinidase (28). Importantly, only a small decrease was noted for the dissociation of 3 from rSAv and succinylated rSAv. Binding of 3 with arginine-modified rSAv showed a somewhat faster dissociation rate. Comparative Tissue Distribution Studies. A tissue distribution study was conducted to compare succinylated rSAv with arginine-modified rSAv. In the study, the proteins were labeled (125I or 131I) and were coinjected into athymic mice so that a direct comparison could be made. Unlike most proteins, direct radioiodination of rSAv provides a radiolabel that is not susceptible to in vivo deiodination (23), presumably because the rSAv is highly resistant to protease degradation. Importantly, use of radioiodination methods that employ conjugates, such as [I*]iodobenzoates (29), was not an attractive alternative for these studies, as a decrease in kidney localization had been previously observed when rSAv was radioiodinated with a p-[I*]]iodobenzoyl labeling moiety (23). Thus,

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Figure 4. Graphs of the data obtained from the biodistribution of (coinjected) arginine-modified [125I]rSAv and succinylated [131I]rSAv in 15 athymic mice. The data obtained for each reagent (radionuclide) is shown in a separate graph for simplicity. Graph 4A shows the tissue distribution of succinylated (50 equiv) [131I]SAv at 4, 24, and 48 h pi. Graph 4B shows the plotted data for arginine-modified [125I]rSAv at 4, 24, and 48 h pi. The mice were placed on biotin-free diets for 7 days prior to the initiation of the experiment. Five animals were used per time point.

methods of radiolabeling that modified the surface amine groups could not be used because that would complicate the interpretation of the effect of arginine modification on the kidney localization. We chose to use athymic mice, to place them on biotinfree diets for 7 days prior to the study, and to administer a total of 100 µg of rSAv to each mouse so that the results would reflect their distribution in the athymic mouse xenograft model used in our pretargeting studies. The results of the biodistribution of succinylated [131I]rSAv and arginine-modified [125I]rSAv at 4, 24, and 48 h pi are shown graphically in Figures 4A and 4B, respectively (full data provided as Tables 2 and 3 in Supplemental Information). While kidney localization at 4 h pi is similar for the two modified rSAvs, kidney retention of radioactivity at 24 and 48 h pi is much higher for argininemodified rSAv. As noted in previous studies (17), the retention of the succinylated rSAv in blood is higher than that of the arginine-modified rSAv. It also appears that there are higher concentrations of succinylated rSAv in the liver and spleen than for the arginine-modified rSAv, but this may be due in part to the higher blood concentration. Varying Levels of Succinylation. An evaluation was conducted to determine how the extent of rSAv modification with succinic anhydride affected kidney localization. In the experiment, 5, 10, 25, and 50 equiv of succinic anhydride were reacted with rSAv. As an initial step in

Figure 5. Digitized images of IEF gels (A and B) showing the rSAv products obtained when modified with various quantities of succinic anhydride. Samples were placed on opposing sides of the gel and run in two different directions to ensure equilibrium was reached. Arrows at bottom of lanes depict direction of sample movement. (A) Lanes 1, 4, and 8: IEF standards (isoelectric calibration kit, broad pI; 3-10) run in opposite direction to application of the adjacent sample. Lanes 2 and 3: rSAv succinylated with 5 equiv of succinic anhydride. Lanes 5 and 6: rSAv succinylated with 10 equiv of succinic anhydride. Lane 7: rSAv which has not been succinylated (control). (B) Lanes 1, 5, and 8: IEF standards (isoelectric calibration kit, broad pI; 3-10) run in opposite direction to application of the adjacent sample. Lane 2: rSAv which has not been succinylated (control). Lanes 3 and 4: rSAv succinylated with 25 equiv of succinic anhydride. Lanes 6 and 7: rSAv succinylated with 50 equiv of succinic anhydride.

the evaluation, the product of each of these reactions was evaluated by IEF. The IEF obtained are shown in Figure 5A and 5B. Modification of rSAv by reaction with 5 equiv of succinic anhydride yielded a major component (pI ∼6.6) and four minor components (pI ∼6.0, 5.8 5.3, 5), but left 50% of the rSAv unmodified (pI ∼7.4-7.8). The modified rSAv from the reaction of 10 equiv of succinic anhydride contained only a trace of unaltered rSAv and a small quantity of the component at pI ∼6.6, but had four major components (pI ∼5.8, 5.3, 5, and 4.7). The modified rSAv from the reaction of 25 equiv had all components in a

Succinylated and Arginine-Modified Streptavidin

Figure 6. Graph of the biodistribution of [125I]rSAv that has been modified with 5, 10, 25, or 50 equiv of succinic anhydride. A control [131I]rSAv was coinjected with each of the preparations, and an example is shown for comparison in the graph. The biodistribution was obtained in 20 athymic mice (five per time point) at 24 h pi.

broad band (pI ∼ 4-5). The modified rSAv obtained in the reaction of 50 equiv of succinic anhydride was very acidic, with all components merging in a broad band at pI ∼3.8-4.5. Biodistributions were determined for each of the rSAv preparations that had been modified with varying amounts of succinic anhydride. In contrast to the direct comparison of 1,2-cyclohexanedione and succinic anhydride modified rSAv, (1) the mice were not placed on biotin free diets, (2) smaller quantities of protein were administered (i.e., 30 µg/mouse), and (3) the distributions were examined at only one time point (i.e., 24 h pi). These changes were made to simplify the study, as it was believed that the conditions in the previous study were not required for this evaluation. The results of the study are shown graphically in Figure 6. The data indicate that the level of succinylation dramatically influences the kidney concentration of streptavidin. Although lower absolute concentrations (%ID/g) were observed in the blood in this study, a similar increase in concentration of succinylated rSAv over the unmodified rSAv was noted. The lower blood concentrations are believed to be due to the smaller amount of protein administered, but may also be due to the fact that the mice were not placed on biotin-free diets prior to conducting the biodistribution, or due to other factors that are not obvious. DISCUSSION

The purpose of this investigation was to compare succinic anhydride-modified rSAv with arginine-modified rSAv, both being potential reagents for use as carriers of radionuclides in “two-step” pretargeting protocols. Although a number of studies have been conducted to evaluate the use of SAv as a carrier of radionuclides in pretargeting protocols (30-36), more recently most pretargeting studies have been focused on using biotin derivatives as carriers of the radionuclide. Our laboratory is particularly interested in the application of R-particle emitting radionuclides, such as At-211 and Bi-213, to therapy of metastatic cancer. At present it appears that the most promising approach to deliver these radionuclides to cancer cells in vivo is to use a monoclonal antibody-based pretargeting protocol. Thus, we have been developing new biotin and SAv pretargeting reagents for this application (37). While we are investigating the use of biotin derivatives as radionuclide carriers in pretargeting, we are also interested in using rSAv as a carrier.

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The rationale for using rSAv as a carrier molecule is based on the fact that it is cleared much more slowly from the blood than biotin derivatives (24), its slow degradation may provide stability to the radiolabel, and its increased blood circulation time may permit injection of smaller quantities of these expensive isotopes. Further, we believe that the toxicity of R-emitting radionuclides that decay while circulating in the blood stream will be minimal due to the short path length of the R particle. Preliminary studies involving the use of monoclonal antibodies labeled with R-emitters for bone marrow conditioning in transplantation seem to support that belief (38). SAv has an inherently high and sustained kidney localization (13,30). This fact could preclude its use as a carrier of radionuclide in pretargeting protocols, so methods of decreasing the kidney localization have been of interest. The ideal rSAv for carrying radionuclides in pretargeting protocols would circulate freely in the blood without localization to tissues other than tumor sites. While kidney localization is dramatically decreased after modification, inspection of the biodistributions of succinylated rSAv (Figure 4A) and arginine-modified rSAv (Figure 4B) indicates that neither of these proteins are ideal carriers. This statement is made, as the concentration of radioactivity in spleen and liver of the mouse model does not diminish as the blood concentration decreases in the distribution of either modified rSAv. Perhaps more troubling is the fact that the kidney concentration of the arginine-modified rSAv does not decrease, suggesting that the radioactivity is trapped there. The biotin binding characteristics of an ideal rSAv molecule for pretargeting is less obvious. It seems likely that the 10-15% faster dissociation rates observed for biotin derivative, 1 or 3, binding with arginine-modified rSAv versus succinylated rSAv would have little effect when short half-lived (R-emitting) radionuclides are used for in vivo applications. A factor that supports use of succinylated rSAv over arginine-modified rSAv is the fact that it is less heterogeneous (by mass spectral analysis, Figure 2B and 2C). While the properties of succinylated rSAv may be only marginally better than arginine-modified rSAv, we will continue to use it in the development of pretargeting protocols for R-emitting radionuclides. To improve succinylated rSAv’s properties, it will need to be optimized. Evaluation of the amount of succinylation required to attain low levels of kidney localization was conducted by studying the products obtained from reaction of 5, 10, 25, and 50 equiv of succinic anhydride. The results obtained from that evaluation clearly indicate that high levels of succinylation are required to eliminate the inherent kidney localization. The IEF (Figures 5A/5B) and biodistributions (Figure 6) of modified rSAv obtained from varying levels of succinylation show that the pI must be lower than 4.5 to diminish the kidney concentration to blood levels. This can be contrasted to the observation that the pI of arginine-modified rSAv is higher (5.2-6), but achieves a similar level of kidney localization at 4 h pi (Figure 4). Although these data provide the basis for using high levels of succinic anhydride to modify the lysine residues, it is interesting to assess the number lysine amines modified in rSAv. The gene for streptavidin encodes for a protein that has eight lysine residues per subunit (39); however, postsecretory modification of SAv (40) provides a “core” protein that is truncated at both the C and N termini, resulting in a protein that has only four lysine residues per subunit (41). Recombinant “core” streptavidin, depending on the

618 Bioconjugate Chem., Vol. 13, No. 3, 2002

termini chosen, may have three or four lysine residues per subunit (42, 43). In the rSAv studied, there are three lysine residues. The mass spectral data indicates that two to three lysines per subunit are modified when 50 equiv of succinic anhydride are reacted for 30 min at room temperature. Our goal for this research was to evaluate whether one method provided a more optimal rSAv for use in pretargeting; however, some interesting observations can be made pertaining to kidney localization of SAv. It has been hypothesized by Wilcheck et al. that the high kidney localization of SAv is due to a surface sequence of amino acids, RYD, which they propose is close enough to the fibronectin binding sequence RGD to bind receptors (14,15). It appeared that a simple test of this hypothesis might be to modify one of the amino acids in the RYD sequence. Arginine was thought to be the best candidate for modification, as it appeared to be the easiest to selectively modify. Fortunately, the amino acid sequences for SAv and rSAv are known (39,42), and crystal structures for many SAv variants are available from the Brookhaven Protein Data Bank. For recombinant SAv, molecular modeling has indicated that one of the four arginine residues (R53) present per subunit is on the surface formed by the amino terminal sequence in residues ∼13-57, and another arginine residue (R84) is on a loop between β-strands in a buried section of the surface (43). Both of these arginine residues appear to be solvent accessible, making it likely that they could be modified. The other two arginine residues (R59 and R103) appear to be in the buried hydrophobic regions. Interestingly, the RYD sequence is present as R59,Y60,D61, of which the aspartic acid residue (D61) appears to be surface-exposed whereas the arginine and tyrosine are not. However, it is possible this sequence could become exposed to the surface at the high pH used in the modification reaction after ionization of the carboxylate of the aspartic acid and the phenol of the tyrosine residues. Therefore, one might predict that 1-3 arginine residues could be modified in the reaction with 1,2cyclohexanedione, and that is what was found. Despite the data presented herein that suggests a modification of a minimum of two lysine residues per subunit are required to alleviate kidney localization, other data seem to indicate that modification of as few as one lysine, or one arginine, per subunit may be all that is required. For example, mass spectral data from the modification of arginine with 1,2-cyclohexanedione that displayed low kidney concentrations had only one arginine residue blocked. This would seem to indicate that modification of one specific arginine (per subunit) is required to diminish kidney localization. Additionally, it was noted that minimal derivatization of rSAv with succinic anhydride (e.g., 5 equiv offered per SAv tetramer) decreased the kidney localization by 50% (Figure 6). In that preparation, the IEF indicated that about 50% of the rSAv subunits were not modified, and the remainder of rSAv subunits may have only a single modification site. Further, previous studies have shown that conjugation of only one [125I]iodobenzoate4 to the SAv tetramer resulted in a decrease in the kidney concentration by 25% (17). These results seem to indicate that a single (solvent accessible) lysine amine per subunit is required for kidney localization. Even if modification of only one

Wilbur et al.

residue is required, complete modification of the four identical lysine or arginine residues in the tetrameric SAv requires an excess of reagents, and it can be expected that other lysine amines or arginine residues would be modified under those conditions. Interestingly, the lysine and arginine modification results are consistent with both the RYD binding hypothesis and another hypothesis put forth by Mogensen and Solling (44). They hypothesized that tubular reabsorption of proteins comes about by binding between a free positive amino or guanidinium group in the protein molecule and a negative site on the tubular cell surface. Our results appear to be consistent with this hypothesis as well. The results obtained indicate that modification of either lysine or an arginine residue greatly diminishes kidney localization over that of rSAv that is not modified. Whether a combination of these functionalities in close proximity is required for kidney localization has not been delineated. These studies have not provided adequate evidence to state that a specific amino acid sequence, such as the RYD sequence, or that a specific amino or guanidinium functionality is required for kidney localization. We previously proposed that altering the charge on the rSAv to be highly negative with succinylation blocked its ability to cross the glomerular wall (17). This possibility has not been substantiated or refuted in these studies. Based on the observation that radioiodinated rSAv is retained in cells when internalized, the fact that the kidney concentration of succinylated rSAv continues to decrease with time may indicate that it is not internalized by the proximal tubular cells in the kidney. Since the kidney concentration in the arginine-modified rSAv does not change with time, it seems likely that it is internalized by the proximal tubular cells. Perhaps the best method to determine whether specific amino acids or amino acid sequences are responsible for kidney localization is to conduct site-directed amino acid substitutions of arginine and/or lysine found at the surface of the rSAv structure and examine the biodistributions of those proteins. We hope to undertake such studies at a later time. Summary. rSAv was modified by reaction with succinic anhydride and 1,2-cyclohexanedione. The reaction of succinic anhydride is selective for conversion of amine functionalities to succinamide derivatives. The modified rSAv obtained after succinylation was found to bind biotin derivatives with nearly the same affinity as unaltered rSAv. The reaction of 1,2-cyclohexanedione is selective for conversion of guanidinium functionalities on arginine residues to produce a mixture of adducts at high pH. From the biotin derivative binding studies, it appears that biotin binding with arginine-modified rSAv has slightly lower affinity. Differences in the in vivo data were not striking. In both reactions, the modified rSAv was found to have relatively low concentration in kidneys of mice. However, the kidney concentration of the succinylated rSAv continued to decrease with time, whereas it remained constant in the arginine-modified rSAv. Additional studies demonstrated that high levels of succinylation were required to obtain the lowest kidney concentrations. The data obtained from these studies indicate that succinylated rSAv has properties that make it a better choice as a radionuclide carrier in pretargeting protocols than arginine-modified rSAv. ACKNOWLEDGMENT

4

It is probable that only one [125I]iodobenzoate group was conjugated per SAv tetramer because of the very small quantity of high specific activity radiolabeled compound used in the conjugation reaction.

We would like to thank personnel at NeoRx Corporation for their discussion of the arginine modification approach. That discussion led to the investigation de-

Succinylated and Arginine-Modified Streptavidin

scribed herein. 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, DOD Prostate Cancer Research Program DAMD17-98-1-8500, and National Institutes of Health RO1 CA76287. Supporting Information Available: Tables containing the complete biodistribution data for arginine-modified and succinylated rSAv (Table 2) and modification of rSAv with varying quantities of succinic anhydride (Table 3). This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Verhaar Langereis, M. J., Zonnenberg, B. A., de Klerk, J. M. H., and Blijham, G. H. (2000) Radioimmunodiagnosis and therapy. Cancer Treatment Rev.. 26, 3-10. (2) Press, O. W. (1999) Radiolabeled antibody therapy of B-cell lymphomas. Seminars Oncol. Oct 26, 58-65. (3) Chatal, J.-F., Peltier, P., Bardie`s, M., Che´tanneau, A., Thedrez, P., Faivre-Chauvet, A., and Gestin, J.-F. (1992) Does Immunoscintigraphy serve clinical needs effectively? Is there a future for radioimmunotherapy? Eur. J. Nucl. Med. 19, 205-213. (4) Buchsbaum, D. J., and Lawrence, T. S. (1991) Tumor Therapy with Radiolabeled Monoclonal Antibodies. Antibodies, Immunoconjugates, Radiopharm. 4, 245-272. (5) Larson, S. M. (1991) Radioimmunology: Imaging and Therapy. Cancer 67, 1253-1260. (6) Hnatowich, D. J. (1990) Antibody Radiolabeling, Problems and Promises. Nucl. Med. Biol. 17, 49-55. (7) Paganelli, G., Riva, P., Deleide, G., Clivio, A., Chiolerio, F., Scassellati, G. A., Malcovati, M., and Siccardi, A. G. (1988) In Vivo Labeling of Biotinylated Monoclonal Antibodies by Radioactive Avidin: A Strategy to Increase Tumor Radiolocalization. Int. J. Cancer 2, 121-125. (8) Chinol, M., Grana, C., Gennari, R., Cremonesi, M., Geraghty, J. G., and Paganelli, G. (2000) Pretargeted Radioimmunotherapy of Cancer, in Radioimmunotherapy of Cancer (P. G. Abrams, and A. R. Fritzberg, Eds.) pp 169-193, Marcel Dekker, New York. (9) Theodore, L. J., Fritzberg, A. R., Schultz, J. E., and Axworthy, D. B. (2000) Evolution of a Pretarget Radioimmunotherapeutic Regimen, in Radioimmunotherapy of Cancer (P. G. Abrams, and A. R. Fritzberg, Eds.) pp 195-221, Marcel Dekker, New York. (10) Axworthy, D. B., Reno, J. M., Hylarides, M. D., Mallett, R. W., Theodore, L. J., Gustavson, L. M., Su, F., Hobson, L. J., Beaumier, P. L., and Fritzberg, A. R. (2000) Cure of human carcinoma xenografts by a single dose of pretargeted yttrium90 with negligible toxicity. Proc. Natl. Acad. Sci. U.S.A. 97, 1802-1807. (11) Press, O. W., Corcoran, M., Subbiah, K., Hamlin, D. K., Wilbur, D. S., Johnson, T., Theodore, L. J., Yau, E., Mallett, R., Meyer, D. L., and Axworthy, D. (2001) A comparative evaluation of conventional and pretargeted radioimmunotherapy of CD20 expressing lymphoma xenografts. Blood 98, 2535-2543. (12) Wilbur, D. S., Hamlin, D. K., Vessella, R. L., Stray, J. E., Buhler, K. R., Stayton, P. S., Klumb, L. A., Pathare, P. M., and Weerawarna, S. A. (1996) Antibody fragments in tumor pretargeting. Evaluation of biotinylated Fab′ colocalization with recombinant streptavidin and avidin. Bioconjugate Chem. 7, 689-702. (13) Wilbur, D. S., Stayton, P. S., To, R., Buhler, K. R., Klumb, L. A., Hamlin, D. K., Stray, J. E., and Vessella, R. L. (1998) Streptavidin in antibody pretargeting. Comparison of a recombinant streptavidin with two streptavidin mutant proteins and two commercially available streptavidin proteins. Bioconjugate Chem. 9, 100-107.

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