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Biotin Reagents for Antibody Pretargeting. 3. Synthesis, Radioiodination, and Evaluation of Biotinylated Starburst Dendrimers D. Scott Wilbur,*,† Pradip M. Pathare,† Donald K. Hamlin,† Kent R. Buhler,‡ and Robert L. Vessella‡ Departments of Radiation Oncology, 2121 North 35th Street, and Urology, University of Washington, Seattle, Washington 98195 . Received May 26, 1998; Revised Manuscript Received September 10, 1998
We are investigating the hypothesis that biotin multimers can be used with streptavidin and monoclonal antibody conjugates in cancer pretargeting protocols to provide a method of increasing the amount of radioactivity bound on cancer cells in patients. As part of that investigation, a series of biotinylated Starburst dendrimers (BSBDs) have been prepared and evaluated in vitro and in vivo. In this study, a new biotinidase-stabilized, water-solubilizing biotinylation reagent was prepared and reacted with Starburst (PAMAM) dendrimers, generations 0, 1, 2, 3, and 4. The reaction conditions employed resulted in perbiotinylation of generation 0 (four biotin moieties conjugated), generation 1 (eight biotin moieties conjugated), generation 2 (16 biotin moieties conjugated), and generation 3 (32 biotin moieties conjugated). With generation 4, incomplete biotinylation was achieved resulting in the largest portion of that BSBD having 51 biotin moieties (of 64 possible) conjugated. The ability of each BSBD to cross-link streptavidin (SAv) was examined in an in vitro assay. In that assay, an assessment was made of the quantity of [125I]SAv bound with polystyrene-bound SAv after treatment with the synthesized BSBDs. All BSBDs cross-linked the polystyrene-bound SAv with [125I]SAv; however, the amount of [125I]SAv bound varied with the different BSBDs. Roughly 1 equiv of [125I]SAv was bound when Starburst dendrimers containing three or four biotin moieties (generation 0) were used. Two equivalents were bound with BSBD generation 1, and 4 equiv were bound with BSBDs generations 2, 3, and 4. To assess the distribution of BSBDs generations 0, 1, and 2 in mice (at 4 h postinjection), a method was developed for radioiodinating them using the NHS ester of p-[125I]iodobenzoate ([125I]PIB). It was found that the radioiodinated BSBDs had low blood concentrations (i.e., 0.13-0.20% ID/g) at the 4 h time point. In fact, most tissues examined had low concentrations of biotinylated dendrimers, except kidney and liver. Kidney had the highest concentration of [125I]labeled BSBDs, and its concentration increased with increasing size and charge of dendrimer (e.g., 8-48% ID/g). On the basis of the increased radioactivity observed in the in vitro assay and the rapid clearance from blood in mice, additional in vivo studies with perbiotinylated Starburst dendrimer, generation 2, are planned.
INTRODUCTION
Reagents containing biotin, avidin, or streptavidin and tumor-specific monoclonal antibodies (mAbs),1 in combination with a variety of radionuclides, are under investigation in an approach to cancer therapy that is termed “pretargeting” (1-3). The pretargeting approach is being investigated as a means of circumventing some of the inherent problems associated with directly radiolabeled * Author to whom correspondence should be addressed. Phone: 206-685-3085. Fax: 206-685-9630. E-mail: dswilbur@ u.washington.edu. † Department of Radiation Oncology. ‡ Department of Urology. 1 Abreviations: BSA, bovine serum albumin; BSBD(s), biotinylated Starburst dendrimer(s); ChT, chloramine-T; cpm, counts per minute; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; G ) 0, 1, 2, 3, or 4, Starburst dendrimer generation 0, 1, 2, 3, or 4; IBz-Trimer, iodobenzoyl-biotin trimer; MALDI-TOF MS, matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry; NCS, N-chlorosuccinimide; PBS, phosphate-buffered saline; r-SAv, recombinant streptavidin; rt, room temperature; (strept)avidin, denotes avidin or streptavidin; SAv, streptavidin; SBD(s), Starburst dendrimer(s); TFP, tetrafluorophenyl; TFP-OH, tetrafluorophenol; TsCl, p-toluenesulfonyl chloride.
mAbs in therapy protocols (4-6). Importantly, in the pretargeting approach, the tumor selective targeting of mAbs is retained, but the delivery of the radionuclide is relegated to another molecule, which in many investigations is either a radiolabeled biotin derivative or a radiolabeled avidin or streptavidin molecule (7-17). The selection of these reagents for the pretargeting approach has been made because of the very high binding avidity of biotin with avidin (18, 19) and streptavidin (20). Tumor localization of the radiolabeled biotin or (strept)avidin is achieved by binding with an antibody conjugate that has been previously localized on tumor cells. Because avidin and streptavidin are composed of four identical subunits, four biotin molecules can bind each protein molecule. This tetrameric binding nature of avidin and streptavidin permits their use in various combinations of mAbs/biotin/(strept)avidin, which are administered in “2-step” or “3-step” pretargeting protocols (6, 21). The introduction of multiple “steps” in tumor targeting provides additional variables for optimizing the pharmacokinetics and distributions of the therapeutic radionuclide not previously obtainable with directly labeled mAbs (5). In addition to improving the pharmacokinetics and distribution of the therapeutic radionuclide, the use of
10.1021/bc980055e CCC: $15.00 © 1998 American Chemical Society Published on Web 10/22/1998
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Wilbur et al.
Figure 1. Schematic representation of biotin multimers cross-linking (strept)avidin with mAb-(S)Av conjugates bound to cancer cell antigens. Amplification (doubling) of the number of binding sites is provided in reaction sequence I by cross-linking tumor bound (strept)avidin with a biotin trimer (B) or biotin tetramer. In reaction I, either the biotin trimer or (strept)avidin molecule can be used as a carrier of the radionuclide. With Starburst dendrimer based biotin multimers (E) more than one (strept)avidin molecule (C) may be added in a single administration as depicted in reaction sequence II. In reaction II, amplification is obtained by having the radiolabel on the (strept)avidin molecule.
biotin/(strept)avidin-based reagents in pretargeting may provide a method for increasing the amount of radionuclide bound to cancer cells in vivo (3, 7, 15). If the number of binding sites for either radiolabeled biotin or radiolabeled (strept)avidin is increased, then it may be possible for more radioactivity to be bound to each cancer cell. One possible method for increasing the biotin/ (strept)avidin-binding sites on cancer cells is to crosslink (strept)avidin with molecules that contain more than two biotin moieties as depicted in Figure 1. For example, a radiolabeled biotin trimer (B in Figure 1) could be used in a multistep protocol that alternates administration of that reagent with (strept)avidin (C in Figure 1) (22). In such a protocol, it might be possible to regenerate an equivalent number of new binding sites (biotin moieties) as there are bound mAbs with each administration cycle. If this occurred, the repetitive cycles could provide a doubling, tripling, and quadrupling of the total amount of radioactivity on the cancer cells in sequential steps. Such a protocol is under investigation in our research group, but it is not an ideal protocol for clinical adaptation as it involves multiple administrations of biotin/ (strept)avidin reagents [and blood clearing agents/ methods (23-28)]. Our desire to maximize the amount of radioactivity deposited on tumor cells in as few steps as possible led to consideration of molecules that contain multiple biotin moieties for cross-linking of streptavidin. Unlike biotin trimers, the use of molecules which contain multiple (i.e., more than four) biotin moieties could lead to the addition of two or more streptavidin molecules in a single step. Although there are many polymeric molecules that could be used as carriers of multiple biotin moieties,2 polyamidoamines (PAMAM) Starburst dendrimers (29-31) were particularly attractive candidates as they are small
spherical compounds, and a variety of sizes (generations)3 is available from commercial sources. We hypothesized that the number of streptavidin molecules bound to an mAb-streptavidin conjugate (F in Figure 1) might be significantly increased in a single step using the biotinylated Starburst dendrimers (E in Figure 1). As an initial evaluation of that hypothesis, an investigation of the synthesis, radioiodination, in vitro binding, and in vivo distribution of biotinylated Starburst dendrimers (BSBDs) has been conducted. In the investigation, a new biotinylation reagent was prepared in which the linking moiety provides increased water solubility and also provides resistance to degradation by the enzyme biotinidase (32-34). Biotinylation of five Starburst dendrimers (SBDs) containing a large variation in the number of biotin moieties per molecule was synthesized. Subsequently, the number of streptavidin (Sav) molecules that can bind with the biotinylated dendrimers was determined by an in vitro assay using streptavidin-coated wells. On the basis of the results of the in vitro studies, three radioiodinated biotinylated dendrimers were prepared. The radiolabeled biotinylated Starburst dendrimers were administered to athymic mice, and their distributions in vivo were determined at 4 h postinjection. The results of our investigation are described herein. 2 For example, polybiotinylated aminodextrans are commercially available from Molecular Probes (Eugene, OR). 3 Starburst Dendrimers of increasing sizes are given the terminology of “generations” as they are built up from “shells”. The generations (abbreviated G) used are 0 (G ) 0; four terminal amines), 1 (G ) 1; eight terminal amines); 2 (G ) 2; 16 terminal amines); 3 (G ) 3; 32 terminal amines); and 4 (G ) 4; 64 terminal amines). Information on the Starburst dendrimers used in this study is provided in Table 1. SBD nomenclature and chemistry are provided in reviews by Tomalia (29-31).
Biotinylated Starburst Dendrimers EXPERIMENTAL PROCEDURES
General. Chemicals purchased from commercial sources were of analytical grade or better and were used without further purification. Solvents for HPLC analysis were obtained as HPLC grade and were filtered (0.2 µm) prior to use. Starburst Dendrimers, d-Biotin; triethylene glycol, 1; tert-butyl acrylate, 2; methylamine, 2,3,5,6tetrafluorophenol; chloramine-T; and most other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI). Recombinant streptavidin (r-SAv) was obtained as previously described (35). BSA was Fraction V (catalog no. 160069) obtained from ICN (Costa Mesa, CA).4 Silica gel chromatography was conducted with 70230 mesh 60 Å silica gel (Aldrich Chemical Co.). Sephadex G-25 (NAP-10) columns were obtained from Pharmacia Biotech AB (Uppsala, Sweden). Avidin and ReactiBind streptavidin-coated polystyrene 96 well plates were obtained from Pierce (Rockford, IL). Melting points were obtained in open capillary tubes on a Mel-Temp II apparatus with a Fluke 51 K/J electronic thermometer and are uncorrected. 1H-NMR data, MS data, and HPLC chromatography were used to assess compound identity and purity (data provided in the Supporting Information). Spectral Analyses. 1H NMR spectra were obtained on either a Bruker AC-200 (200 MHz) or a Bruker AC500 (500 MHz) instrument. Proton chemical shifts are expressed as parts per million using tetramethylsilane as the internal standard (δ ) 0.0 ppm). IR data were obtained on a Perkin-Elmer 1420 infrared spectrophotometer. Mass spectral data for the compounds of low molecular weight (i.e., 60 Å length (biotin carbonyl carbon to second biotin carbonyl carbon) to bind at opposite faces of SAv. The estimated through bond distances between any two biotin moieties in the SBDs G ) 1-4 (Table 1) appear to be adequate for more than two biotin moieties to bind with a single SAv molecule. Irrespective of the explanation for the results obtained, there appears to be no advantages for our application to use BSBDs larger than generation 2. Even though a smaller number of SAv molecules were attached to the immobilized BSBDs than might be predicted, substantial amplification of the amount of radioactivity bound to the SAv-coated plates was obtained. We have previously shown that in a single addition, cross-linking with biotin trimers in the same assay system resulted in 4-6 pmol of [125I]SAv bound/ well. Further, it was demonstrated that a doubling of that quantity (10-12 pmol) of [125I]SAv was obtained in four alternating cycles of reagent additions (22). Those results were reproduced in this study with biotin trimer 29 and iodobenzoyl-biotin trimer 21b (Figure 2). However, much larger quantities (16-30 pmol) of [125I]SAv were bound when biotinylated SBDs 15, 17, and 19 were used. Indeed, with one addition of the biotin multimers 15, 17, or 19, there was a 300% increase of [125I]SAv bound over that of a single addition of the biotin trimers 29 or 21b, and a 40+% greater quantity bound than obtained previously with four alternating administrations of the biotin trimers. The in vitro results support the potential for amplification of tumor targeting of radioactivity using the biotin multimers and radiolabeled SAv. As our ultimate goal is to use the BSBDs for in vivo application to tumor pretargeting of radionuclides for cancer therapy, it is important to evaluate the in vivo distribution and pharmacokinetics of the BSBDs. We have previously shown that a variety of biotin trimers cross-linked SAv in the coated wells (22), but we had not conducted in vivo distribution studies with the biotin trimers. As we are interested in the development of radiolabeled biotin trimers as carriers of radionuclides, [125I]21b was included in the in vivo evaluation. The BSBD [125I]23, which has 15 biotin moieties, appeared to be the best candidate for in vivo amplification of binding sites, but the intermediate size of BSBD [125I]22 made its in vivo distribution of interest as well. Although it is envisioned that the perbiotinylated SBDs G ) 1 or 2 will not be radiolabeled for in vivo use, it was necessary to radiolabel them to assess their biodistribution. To utilize the data obtained, it must be assumed that the radioiodinated versions of the biotinylated dendrimers, [125I]22 and [125I]23, will have distribution similar to the perbiotinylated SBDs 13 and 15 (respectively). In this preliminary investigation, we felt that it would be adequate to obtain comparative information about the tissue distribution of the three BSBDs ([125I]21b, [125I]22, and [125I]23) in athymic mice7 at one time point (i.e., 4 h postinjection). The limited biodistribution study was designed to determine if additional, more detailed dis6 Measurements of distances in biotin derivatives were obtained from the computer program ChemDraw3D (CambridgeSoft Corporation, Cambridge, MA) after structural and energy minimization of fully extended, planar conformations of the compounds. 7 Athymic mice (BALB/c nu/nu) were used in this investigation so that the results can be compared with planned studies involving human tumor xenografts in athymic mice.
Bioconjugate Chem., Vol. 9, No. 6, 1998 823
tributions were warranted. The results of the investigation are shown in Table 3. Interestingly, the concentrations of radiolabeled BSBDs in blood at 4 h postinjection are nearly the same. Differences in distribution between radiolabeled BSBDs are minor except for the kidney and liver concentrations. In previous distributions of radioiodinated biotin monomers in mice, blood clearance was similar at 4 h to that found for [125I]21b, but quite high intestine concentrations were observed (unreported results). In this investigation, intestinal concentration of radioactivity was not particularly high for the biotin trimer [125I]21b, but the kidney concentration is troubling. The high kidney concentration is problematic for a carrier of radioactivity, so chemical modification of the biotin trimer is being investigated to make it pass through the kidney more rapidly. Contrary to the biotin trimer, the high concentrations of [125I]22 and [125I]23 in the kidney and liver at 4 h postinjection may not present a problem for application to cross-linking SAv on cancer cells, as they will not be used as carriers of radionuclides. In Summary. In this study, Starburst dendrimers, 10 (G ) 0; four amines), 12 (G)1; 8 amines), 14 (G ) 2; 16 amines), 16 (G ) 3; 32 amines), and 18 (G ) 4; 64 amines), were reacted with a new water solubilized, biotinidase stabilized biotinylation reagent, 9, to prepare polybiotinylated dendrimers for in vivo application. Data from an in vitro binding assay indicates that the biotinylated Starburst dendrimer 11, which has four biotin moieties, binds one 125I-labeled SAv molecule in addition to a SAv molecule bound to the polystyrene well. Biotinylated Starburst dendrimer 13, which has eight biotin moieties, binds two additional [125I]SAv molecules, and 15, which has 16 biotin moieties, binds four additional [125I]SAv molecules. Larger biotinylated Starburst dendrimers 17 and 19 were similar to 15 in that they only bound four additional SAv molecules. Biodistributions of radioiodinated polybiotin dendrimers in mice indicated that they were cleared from blood by 4 h, but that relatively high kidney and liver concentrations were noted at that time. A substantial increase in kidney localization or retention was noted as the polybiotin compounds increased in size and charge. The results presented herein suggest that further investigation of the concept of amplifying the amount of radiolabeled SAv that will bind to a tumor through the use of BSBDs is warranted. Animal studies to investigate this are planned. ACKNOWLEDGMENT
We thank Dr. Ross Lawrence (Department of Medicinal Chemistry, University of Washington) and Santosh Kumar (Department of Biochemistry, University of Washington) for efforts in obtaining mass spectral data. We thank Dr. Patrick Stayton and Dr. Richard To for providing r-SAv used in the studies, and for their helpful comments on the manuscript. 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 DE-FG06-95ER62029, and the Richard M. Lucas Foundation. Supporting Information Available: Complete structures for the biotinylated dendrimers are provided along with HPLC chromatograms, NMR spectra, and mass spectra of all new compounds (4, 5, 7, 9, 11, 13, 15, 17, 19, 21a, and 21b). An HPLC chromatogram of biotin derivative 8, which was not isolated, is also included (41
824 Bioconjugate Chem., Vol. 9, No. 6, 1998
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BC980055E
