146
Bioconjugate Chem. 1999, 10, 146−149
Biotin-Conjugated Polychelating Agent Vladimir P. Torchilin* Center for Imaging and Pharmaceutical Research, Department of Radiology, Massachusetts General Hospital and Harvard Medical School, 149 13th Street, Charlestown, Massachusetts 02129. Received May 27, 1998
A procedure is described for preparing a polychelating agent-biotin conjugate through the interaction of biotin-maleimide with poly-L-lysine modified with multiple residues of diethylenetriaminepentaacetic acid and containing amino-terminal pyridylthio-propionate group. The conjugate can be easily loaded with multiple metal atoms, such as 111In, and preserves its ability to specifically interact with avidin (as avidin-agarose). Polychelate-biotin conjugate may be used for signal amplification in visualization and analytical protocols involving the application of the avidin-biotin system.
The avidin-biotin system is well studied and used for different biochemical and biomedical applications including analytical ones, for review see ref 1. Its application is based on the ability of avidin macromolecule to bind four biotin molecules with extremely high affinity (Kd ≈ 10-15 M). In particular, the authors of ref 1 mention the application of avidin-biotin complex in affinity chromatography, affinity cytochemistry, cell cytometry, blotting technology, immunoassay, histopathology staining, gene probes, and bioaffinity sensors. Avidin-biotin complex is also being used for targeted delivery of diagnostic and therapeutic agents. For the latter purpose, a ligand is modified either with avidin or with biotin and is then allowed to accumulate in a target area. Then, the pharmaceutical agent is administered as a conjugate with biotin (if avidin is accumulated in the target) or with avidin (if the target contains biotin) and concentrates locally in the desired area due to the fast formation of avidin-biotin complex. Since the in vivo stability of biotin association with various biological (macro) molecules was repeatedly confirmed (see, for example, refs 2 and 3 and references therein), the approach is currently finding growing application. Recently, the (strept) avidin-biotin system was also used for the visualization of various pathological conditions including tumors (4, 5). The approach is based on the observation that infected, inflamed, necrotic, and malignant areas are able to nonspecifically accumulate large protein molecules, such as immunoglobulins or albumin (6, 7) or even nanoparticulates (8) via the mechanism of impaired filtration. It was suggested to use (strept) avidin itself or (strept) avidin-antibody conjugate as a tumor-accumulating substance, and after its preliminary injection and accumulation, to target (i.e., to visualize) the tumor with a biotin-associated label (911). Different heavy metal ions serve usually as those labels, such as 111In and 99mTc (for γ-immunoscintigraphic visualization); Gd and Mn (for magnetic resonance imaging), or 186,188Re and 90Y (for tumor radiotherapy) (10). Whenever biotin has to be modified with a reporter metal for in vitro or in vivo application, to couple metals with biotin, the latter is first chemically modified with a * To whom correspondence should be addressed. Current address: Department of Pharmaceutical Sciences, Northeastern University, Mugar Building 312, 360 Huntington Avenue, Boston, MA 02115.
metal-chelating moiety (11). However, the insufficient ability to load biotin with a metal might be considered as one of the potential limitations of the proposed scheme. All techniques described so far involved the use of biotin carrying just a single chelating group, i.e. capable of binding just one metal atom, which results in the insufficient signal multiplication from biotin in both, in vitro and in vivo analytical (imaging) procedures. Thus, in vivo it should result in relatively low label accumulation in the target (tumor), especially when the size of the target is small and/or the quantity of avidin in the target is low. This makes γ- and MR-imaging as well as radiotherapy difficult enough, since the efficacy of all in vivo protocols depend on the quantity of a label delivered to the target. The sensitivity of various analytical in vitro protocols involving avidin-biotin application also might suffer because of insufficient biotin load with tracers. This report describes an attempt to increase biotin load with a marker metal. With this in mind, the scheme was developed of biotin modification with single-terminus reactive chelating polymers containing multiple chelating groups and able to chelate firmly multiple metal atoms (their exact number in each particulate case can be controlled by the length of a chelating polymer). Such polymers were developed by us earlier for the modification of monoclonal antibodies used for γ-immunodiagnostics (12-14), see also ref 15 for review. Materials. Biotin-maleimide [N-biotinoyl-N′-(6-maleimidohexanoyl)hydrazide, BMA], N-(carbobenzyloxy)poly-DL-lysine (CBZ-PLL; MW 4000), diethylenetriaminepentaacetic acid cyclic anhydride (caDTPA), dithiothreitol (DTT), N-succinimidyl 3-(pyridyldithio) propionate (SPDP), guanidine chloride, and all solvents and buffer components were obtained from Sigma Chemical Co., St. Louis, Mo.1 Prepacked 1 mL avidin-agarose columns (ImmunoPure AffinityPak) were acquired from Pierce (Rockford, IL). 1Abbreviations: BMA, N-biotinoyl-N′-(6-maleimidohexanoyl)hydrazide; PLL, poly-DL-lysine; CBZ-PLL, N-(carbobenzyloxy)poly-DL-lysine; caDTPA, diethylenetriamine pentaacetic acid cyclic anhydride; DTT, dithiothreitol; SPDP, N-succinimidyl 3-(pyridyldithio)propionate; DTPA-PLL-PDP, N-terminal-activated chelating polymer; DTPA-PLL-SH, chelating polymer with free terminal SH-group; DTPA-PLL-biotin, conjugate of chelating polymer with biotin; DTPA-biotin, conjugate of monomeric DTPA with biotin; HBS, HEPES-buffered saline.
10.1021/bc9800567 CCC: $18.00 © 1999 American Chemical Society Published on Web 12/24/1998
Biotin-Conjugated Polychelating Agent
Bioconjugate Chem., Vol. 10, No. 1, 1999 147
Figure 1. The scheme of DTPA-PLL-biotin synthesis. Starting DTPA-PLL-PDP was prepared as in ref 13.
Synthesis. N-Terminal-activated chelating polymer DTPA-PLL-PDP was prepared as in ref 14 by modification of the unprotected terminal amino group in CBZPLL with SPDP, subsequent deprotection of CBZ-PLLPDP with HBr/CH3COOH, and interacting free amino groups of PLL-PDP with caDTPA. For this purpose, 3.5 µmol of SPDP was reacted with 0.8 µmol of CBZ-PLL (MW 4000) in 350 µL of DMFA in the presence of 0.14 µL of triethylamine at 4 °C overnight. The product was precipitated with water and CBZ deprotected with 250 µL of 36% HBr in acetic acid. The resulting PLL-PDP was precipitated with diethyl ether and freeze-dried. DTPA residues were incorporated into PLL-PDP by reacting the latter with 50-fold molar excess of caDTPA at pH 8.2. In order to diminish possible cross-linking of PLL chains by bifunctional caDTPA, mixed DTPA anhydride can be used instead of caDTPA (16). However, HPLC analysis of DTPA-PLL-PDP obtained by both protocols provides similar patterns demonstrating the absence of olygomeric structures. As was demonstrated in our previous reports using the titration of remaining free amino groups with trinitrobenzene sulfonic acid of direct binding of 111In (12-14, 17), in such conditions 1520 DTPA residues can be reproducibly incorporated into a single chain of DTPA-PLL(4000)-PDP. To derivatize biotin with DTPA-PLL, DTPA-PLL-PDP (10 mg/mL in HEPES buffered saline, HBS, pH 7.0) was first reduced with 5 mM DTT in HBS for 2 h at room temperature. The product was purified from low-molecular-weight reactants by centrifugation on Sephadex G-10 minicolumn (Pharmacia). The resulting chelating polymer with free terminal SH-group was conjugated with BMA. For this purpose, 100 µg of DTPA-PLL-SH was incubated with 6 µg of BMA (added as 10% solution in DMSO), which corresponds to molar ratio DTPA-PLLSH:BMA ca. 2:1, in 0.5 mL of HBS, pH 7.4, for 1 h at room temperature. The resulting DTPA-PLL-biotin (mixed with nonreacted DTPA-PLL-SH and BMA) was separated from low-molecular-weight reactants, including BMA, by centrifugation on Sephadex G-10 minicolumn. The synthesis scheme is presented in Figure 1. DTPA-PLL-biotin (as a mixture with nonreacted DTPAPLL-SH) and control DTPA-PLL-SH (same molar quantities as DTPA-PLL) were labeled with 111In via the transchelation mechanism. For this purpose, 150 µL of 0.1 M citrate, pH 5.3, was added to 100 µg of each polymer in 0.5 mL of HBS, then polymer solutions were supplemented with ca. 300 µCi of 111In (as 111InCl3 in HCl and
Figure 2. Chromatography of 111In-labeled DTPA-PLL-biotin and control DTPA-PLL on avidin-agarose without (A) and with (B) column presaturation with free biotin. Column volume, 5 mL; samples were applied in 0.5 mL of HBS, pH 7.4 (other details in the text).
NEN) and incubated for 1 h at room temperature. Free was separated from 111In-labeled polymers by centrifugation on Sephadex G-10 mincolumn. 111In
RESULTS AND DISCUSSION
Sephadex G-10 chromatography of 111In-labeled samples demonstrated that both the eluate with DTPA-PLLBiotin and eluate with DTPA-PLL-SH contained ca. 250 µCi of 111In radioactivity (of ca. 300 µCi added to each sample). It proves efficient and similar labeling of chelates in both polymers. To check the efficacy of biotin derivatization with DTPA-PLL, a gel-chromatography of both 111In-labeled DTPA-PLL-Biotin and control DTPA-PLL-SH on avidineagarose column equilibrated with HBS was performed. The results of this experiment are presented in Figure 2A. Upon washing column with HBS, all applied polymerbound radioactivity was eluted in case of control chelating polymer, DTPA-PLL-SH. However, only 60% of applied radioactivity was eluted in case of labeled DTPA-PLLBiotin. Forty percent of applied radioactivity remained on the column. This avidin-agarose-bound radioactivity
148 Bioconjugate Chem., Vol. 10, No. 1, 1999
Figure 3. Radioactivity (111In) binding with avidin-agarose via DTPA-biotin (1) and DTPA-PLL-Biotin (2). For conditions, see Figure 2.
cannot be eluted by washing the column with 1 M NaCI (which excludes any nonspecific interactions, such as electrostatic ones), but was eluted as a broad peak with 8 M guanidine chloride, which is known to slowly dissociate avidin-biotin complex (18). However, presaturation of the avidine-agarose column with pure nonlabeled biotin results in identical elution pattern for both polymers: HBS is able to elute all applied radioactivity for both, DTPA-PLL-biotin and control DTPA-PLL-SH (Figure 2B). It clearly proves that the reaction mixture after DTPA-PLL-biotin synthesis, together with nonbiotinylated DTPA-PLL, contains also polychelate-conjugated biotin which preserves its ability to specifically interact with avidin with very high affinity since, like in case of the native biotin-to-avidin interaction, only 8 M guanidine chloride is able to slowly dissociate the complex formed. Probably, negatively charged polychelator forms rigid rod-like “tail” attached to biotin “head” and does not create any steric hindrances for the biotin-to-avidin interaction. Since in the reaction mixture, DTPA-PLLSH:BMA molar ratio was 2:1 and avidine-agarose adsorbs ca. 40% of applied reaction mixture (following the radioactivity data), we can easily calculate that the coupling yield of BMA and DTPA-PLL-SH is ca. 80% relative to BMA. Thus, even in the case of relatively low-molecularweight PLL, it is possible to couple a PLL tail containing 15-20 DTPA residues (it means the same number of diagnostic or therapeutic metal atoms) to a single biotin molecule. To prove the practical significance of polychelate-biotin and its superiority over traditional single chelate-modified biotin, we compared the quantity of radioactivity which can be delivered to the avidin-agarose column by 111In-loaded DTPA-biotin (was purchased from Sigma as DTPA-R,ω-bis-biocytinamide) and DTPA-PLL-Biotin under similar conditions. Both reagents, taken in similar quantities as biotin, were incubated with the same quantity of 111In in the same conditions, and reaction mixtures were directly applied onto identical avidinagarose minicolumns. After washing columns with HBS (to remove all components containing no biotin moiety), the radioactivity remaining on both columns (i.e., attached to avidin via biotin residues) was measured. The results presented in Figure 3 clearly shows that DTPAPLL-biotin is able to deliver to avidin-containing matrix almost 15 times more radioactivity than DTPA-biotin (which corresponds well with the presence of ca. 15 DTPA groups in DTPA-PLL-biotin compared with a single DTPA group in routine DTPA-biotin). Thus, the possibility of increasing loading of biotin with metal (radio)isotopes via chelating polymers is demonstrated. Chelate-PLL-biotinyl conjugates (with various
Torchilin
chelates) can be used to amplify the signal from avidinbound biotin in various analytical systems in vitro and to enhance a diagnostic and/or therapeutic metal targeting to a preaccumulated avidin or avidinylated molecules in vivo. The medical application of this approach might be especially attractive, and one can speculate that the local metal concentration can be achieved suitable for γ-imaging, radiotherapy, and MR diagnostics of avidincontaining targets (tumors, infarcts, necrotic tissues etc.). Sure, additional in vivo studies have to be performed to investigate the pharmacokinetics of biotin-polychelate compared to biotin-monochelate (in particular liver uptake which might be higher for biotin-polychelate if the presence of DTPA residues will not compensate sufficiently positive charge of PLL) before making a more definite conclusion concerning the clinical (diagnostic) applicability of a new conjugate. However, the benefit of using biotin-polychelate conjugates is evident for in vitro analytical biochemistry, where it might be successfully applied as an amplification system in analytical procedures involving the use of the avidin-biotin system. LITERATURE CITED (1) Wilchek, M., and Bayer, E. A. (1988) The avidin-biotin comples in bioanalytical applications. Anal. Biochem. 171, 1-32. (2) Paganelli, G., Samuel, A., Magnani, P., Siccardi, A., and Fazio, F. (1995) Potential of the avidin-biotin system for diagnostic application. in Handbook on Targeted Delivery of Imaging Agents (V. Torchilin, Ed.) pp 289-303, CRC Press, Boca Raton, FL. (3) Jeong, J. M., Saga, T., Lee, J., and Koh, C.-S. (1995) Avidinbiotin system for targeting metastases: basic aspects. in Handbook on Targeted Delivery of Imaging Agents (V. Torchilin, Ed.) pp 305-319, CRC Press, Boca Raton, FL. (4) Hnatowich, D. J., Virzi, F., and Rusckowski, M. (19897) Investigation of avidin and biotin for imaging applications. J. Nucl. Med. 28, 1294-1302. (5) Rusckowski, M., Fritz, B., and Hnatowich, D. J. (1992) Localization of infection using streptavidin and biotin: an alternative to nonspecific polyclonal immunoglobulin. J. Nucl. Med. 33, 1810-1815. (6) Rubin, R. H., Fischman, A. J., Needleman, M., Wilkinson, R., Callahan, R. J., Khaw, B.A., Hansen, W. P., Kramer, R. B., and Strauss, H. W. (1989) Radiolabeled, nonspecific polyclonal human immunoglobulin in the detection of focal inflammation by scintigraphy: comparison with gallium-67citrate and technitium-99m-labeled albumin. J. Nucl. Med. 30, 385-389. (7) Goh, A. S. W., Aw, S. E., Sundram, F. X., Ang, E. S., Goh, S. K., and Leong, K. H. (1990) Imaging of focal inflammation with 99m-Tc-labeled human polyclonal immunoglobulin G. Nucl. Med. Commun. 11, 843-856. (8) Palmer, T. N., Caride, V. J., Caldecourt, M. A., Twicjler, J., and Abdullah, V. (1984) The mechanism of liposome accumulation in infarction. Biochim. Biophys. Acta 797, 363368. (9) Kalofonos, H. P., Rusckowski, M., Siebecker, D. A., Sivolapenko, G. B., Snook, D., Lavender, J. P., Epenetos, A. A., and Hnatowich, D. J. (1990) Imaging of tumor in patients with indium-111-labeled biotin and streptavidin-conjugated antibodies: preliminary communication. J Nucl. Med. 31, 17911796. (10) Antibodies in Radiodiagnosis and Therapy (1989) (Zalutsky, M. R., Ed.) CRC Press, Boca Raton, FL. (11) Virzi, F., Fritz, B., Rusckowski, M., Gionet, M., Misra, H., and Hnatowich, D. J. (1991) New In-111-labeled biotin derivatives for improved immunotargeting. Nucl. Med. Biol. 18, 719-726. (12) Torchilin, V. P., Klibanov, A. L., Nossiff, N. D., Slinkin, M. A., Strauss, H. W., Haber, E., Smirnov, V. N., and Khaw, B. A. (1987) Monoclonal antibody modification with chelatelinded high-molecular-weight polymers: major increase in
Biotin-Conjugated Polychelating Agent polyvalent cation binding without loss of antigen binding. Hybridoma 6, 229-240. (13) Slinkin, M. A., Klibanov, A. L., and Torchilin, V. P. (1991) Terminal-modified polylysine-based chelating polymers: highly efficient coupling to antibody with minimal loss in immunoreactivity. Bioconjugate Chem. 2, 342-348. (14) Torchilin, V. P., Trubetskoy, V. S., Narula, J., Khaw, B. A., Klibanov, A. L., and Slinkin, M.A. (1993) Chelating polymer modified monoclonal antibodies for radioimmunodiagnostics and radioimmunotherapy. J. Controlled Release 24, 111-118. (15) Torchilin, V. P., and Klibanov, A. L. (1991) The antibodylinked chelating polymers for nuclear therapy and diagnostics. CRC Crit. Rev. Ther. Drug Carrier Syst. 7, 275-308.
Bioconjugate Chem., Vol. 10, No. 1, 1999 149 (16) Krejcarek, G.E., and Tucker, K.L. (1977) Covalent attachment of chelating groups to macromolecules. Biochem. Biophys. Res. Commun. 77, 581-584. (17) Trubetskoy, V. S., Narula, J., Khaw, B. A., and Torchilin, V. P. (1993) Chemicallly optimized antimyosin Fab conjugates with chelating polymers: importance of the nature of the protein-polymer single site covalent bond for biodistribution and infarct localization. Bioconjugate Chem. 4, 251-255. (18) Green, N. M., and Toms, E. J. (1972) The dissociation of avidin-biotin complexes by guanidinium chloride. Biochem. J. 130, 707-711.
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