Pretargeted radioimmunotherapy in tumored mice using an in vivo 212Pb/212Bi generator
Introduction
The potential use of antibodies that recognize and selectively bind with high affinity to antigens present on tumor cells has been the basis of a great deal of work to deliver radiation to tumors for radioimmunotherapy [1]. A major limitation of this approach has been a mismatch of the properties of radioactivity with the tumor-targeting processes of the large antibody proteins. Antibody proteins are slowly taken up by tumors as well as are slowly disappearing from the circulation. These properties result in irradiation of radiosensitive marrow cells during the extended circulation time as well as decay losses of the radionuclide during the time required for tumor localization.
Antibody pretargeting [2], [3] is a process that addresses the limitations of antibodies in delivery of radiation to tumors. The process [4], which we have investigated, involves an initial step of tumor targeting of an antibody with a high-affinity acceptor such as streptavidin attached. This allows the slow targeting step to be done with a material that poses no radiotoxic effect. The intact immunoglobulin also provides for the maximum fraction of the ID to be localized at the tumor. After time for tumor uptake, about 20 h in mice or 48 h in patients, a clearing agent, synthetic dendrimeric galactose-biotin, is given to remove the remaining circulating antibody–streptavidin. In the final step, the radioactivity is given as a small molecule, radiobiotin, that binds with very high affinity to the pretargeted acceptor, streptavidin (1015/M affinity). The tumor uptake occurs within minutes, while nontargeted radioactivity is rapidly removed by kidneys via the filtration route. The pretargeting system has been extensively studied in animals and in Phase I/II studies [5] in patients using the β-emitting radiotherapy nuclide 90Y.
α Particles are 4He ions, approximately 8000 times larger than β-particle electrons, and when emitted, result in high linear energy transfer (LET) of around 100 keV/μm compared to β particles such as 90Y, which have a LET of 0.2 keV/μm [6], [7]. These physical characteristics result in radiation properties that have several advantages compared to β particles for targeted radiotherapy in appropriate clinical situations. The high LET results in a small number of targeted α's required to kill a cell, on the order of 10 to 30 with fewer needed if internalized in cells [8]. Effective cell killing is independent of oxygenation levels, dose rate as achievable with intravenous administration (at or above 5 rad/h) or repair capability. Low penetration, on the order of several cell diameters, provides efficient killing of isolated cells, cell clusters or micrometastases, thus, complementing β emitters that require crossfire field effects and are advantageously used in larger tumors. Finally, there is the potential for lower toxicity in circulation due to the short range. Thus, α particle emitters may be advantageous in clinical applications such as micrometastases that limit the ultimate ability to control or cure cancer [9].
Three α emitters have had extensive study and are candidates for development: 211At with a 7.2-h half-life, 212Bi with a 1.06-h half-life and 213Bi with a 46-min half-life. We have selected the 212Pb/212Bi system [10] because it provides an in vivo generator supply of the 212Bi from the 10.6-h half-life 212Pb. The longer lived 212Pb allows time for tumor targeting of the α emitter 212Bi. Key to its use, however, is the ability to form a stable chelate of 212Pb that controls the in vivo localization through the decay of the 212Bi. Works by Ruegg et al. [11], Junghans et al. [12] and Gansow et al. [13] have demonstrated that the cyclic polyamino acid chelating agent DOTA forms stable chelates with both Pb and Bi.
In theory, the decay of 212Pb should not present a problem in retention of the 212Bi. Conservation principles dictate that the recoil energy of the Bi nucleus is only about 0.5 eV. This is not sufficient to break a chemical bond, which requires approximately 10 eV. However, the γ-ray emitted when 212Pb decays is internally converted over 30% of the time. The resulting cascade of conversion electrons makes the metal–chelate complex unstable. When the 212Pb decays to 212Bi, about two thirds remains as a DOTA chelate, and the other third goes through the decay pathway that results in conversion electrons and “dechelation” of the 212Bi during the process [14]. Loss of 212Bi and its release as free bismuth results in kidney localization and was a major concern for the use of this system.
In this study, we reported the in vitro stability of 212Pb/212Bi-DOTA-biotin and their γ-emitting analogues, 203Pb and 205Bi-DOTA-biotin; biodistribution of 203Pb-DOTA-biotin and 205Bi-DOTA-biotin in animals following the pretargeting protocol; and comparison with that of 212Pb and 212Bi isotopes. We report the dosimetry estimates of targeting 212Pb in mouse xenograft model. The kidney dose by following 212Bi activity after dechelation from 212Pb-DOTA-biotin was also reported.
Section snippets
Radionuclides
203Pb and 205/206Bi (for simplicity, the bismuth radioisotope mixture will hereafter be referred to only as 205Bi) were obtained as chloride forms from Nordion International, Vancouver, BC, and Crocker National laboratory, Davis, CA, respectively. Both radionuclides were received with certificates of radionuclidic purity from the manufacturers and were used directly without further purification. 212Pb/212Bi was obtained by eluting a 224Ra/212Pb generator (Argonne National Laboratory, Argonne,
In vitro stability
The results of in vitro stability of 203Pb and 205Bi-DOTA-biotin by avidin beads assay are compared with 111In-DOTA-biotin and are shown in Table 1. Both Pb-DOTA-biotin and Bi-DOTA-biotin radiochelates were stable for at least 4 days in the different challenging solutions including PBS buffer, 10 mM DTPA and serum. In the case of 212Pb/212Bi, we observed greater than 30% free 212Bi 4 h after we labeled 212Pb-DOTA-biotin by both HPLC and the avidin beads biotin binding assay as shown in Table 2.
Urinary metabolite analysis
Discussion
The quantitative labeling of macrocyclic DOTA-biotin ligand with Pb and Bi isotopes was achieved at elevated temperature with the conditions similar to those used for 111In and 90Y [16]. The in vitro stability study showed that there was very little activity released from the DOTA chelate over 4 days in both 203Pb and 205Bi, which is also very similar to the stability of 111In-DOTA-biotin. Different stability was seen using 212Pb; the 212Pb-DOTA-biotin was stable initially, but there was >30%
Acknowledgment
The funding for this work was provided by the National Cancer Institute SBIR (grant 1R43CA71221-1A1). This work was also performed under the auspices of the U.S. Dept. of Energy, Office of Science, Office of Biological and Environmental Research under contract W-7405-ENG36.
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Current address: Bristol-Myers-Squibb Medical Imaging, North Billerica, MA 01862, USA.
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Current address: Shin Nippon Biomedical Laboratories, Everett, WA, USA.
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