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From: Cancer Drug Discovery and Development:
Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ
5
INTRODUCTION
The role of monoclonal antibodies (MAbs) in the treatment of malignancies has increased dramatically over the past decade. Three unlabeled MAbs—
rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, CA, and Genentech, Inc., South San Francisco, CA), trastuzumab (Herceptin; Genentech, Inc., South San Francisco, CA), and alemtuzumab (Campath-1H; Burroughs Wellcome, United Kingdom)—have been approved by the US Food and Drug Administra- tion (FDA) for the treatment of lymphoma, breast cancer, and chronic lympho- cytic leukemia, respectively. These and other unlabeled MAbs kill tumor cells
Targeted F-Particle Therapy
A Rational Approach to Drug Development in Hematological Diseases and Solid Tumors
John M. Burke, MD
David A. Scheinberg, MD, PhD and Joseph G. Jurcic, MD
CONTENTS
INTRODUCTION
RATIONALE FOR TARGETEDF-PARTICLE THERAPY
MECHANISMS OF RADIATION-INDUCED CELL DEATH
SELECTEDF-PARTICLE-EMITTING RADIOISOTOPES
RADIOLABELING
DOSIMETRY
PRECLINICAL AND CLINICAL STUDIES
POTENTIAL TOXICITIES
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by mediating complement-dependent cytotoxicity and antibody-dependent cel- lular cytotoxicity (1) and by directly causing apoptosis (2,3).
Despite these clinical successes, many native antibodies have only weak anti- tumor effects. To overcome this limitation, MAbs have been conjugated to che- motherapeutic agents and to toxins, such as Pseudomonas exotoxin, ricin, gelonin, and diphtheria toxin. For example, gemtuzumab ozogamicin (Mylotarg; Wyeth Laboratories, Philadelphia, PA) consists of an anti-CD33 antibody conjugated to the antitumor antibiotic calicheamicin; it has activity against CD33-positive acute myeloid leukemia and was approved for clinical use by the FDA in 2000 (4).
Another strategy to increase the antitumor activity of MAbs is radioimmu- notherapy, in which antibodies act as carrier molecules to deliver radioisotopes to target cells, where the emitted F- or G-particles damage DNA, resulting in cell death. To date, the only radioimmunoconjugate approved by the FDA for clinical use is yttrium-90 (90Y) ibritumomab tiuxetan (Zevalin; IDEC Pharmaceuticals, San Diego, CA) for the treatment of patients with relapsed or refractory follicular or transformed non-Hodgkin’s lymphoma (5).
Most radioisotopes used in clinical trials have been G-particle emitters. G- particles are negatively charged particles equivalent to electrons that are emitted from the nucleus of radioisotopes. They have a relatively long range in tissue (several millimeters) and low energies (approx 300–2000 keV). Because of these physical properties, approx 10,000 G-particles traversing a cell nucleus are re- quired to kill a target cell. Examples of G-emitters used in clinical radioimmu- notherapy trials include iodine-131 (131I),90Y, and rhenium-188 (188Re).
Despite the predominant use of G-emitters in radioimmunotherapy trials, investigators have recognized the potential advantages of using F-particle emit- ters for years. F-particles are positively charged helium nuclei with a shorter range (50–80 (μm) and higher energy (5000–8000 keV) than G-particles. Ex- amples of F-particle emitters under investigation include astatine-211 (211At), bismuth-212 (212Bi), bismuth-213 (213Bi), and actinium-225 (225Ac). In this chapter, we will review the characteristics of various isotopes for the targeted F- particle therapy of cancer, issues in radiolabeling and dosimetry, and recent pre- clinical and clinical studies. The reader is also referred to other published reviews on this subject (6–8).
RATIONALE FOR TARGETED F-PARTICLE THERAPY The different physical properties of F- and G-particles confer theoretical advantages and disadvantages to each type of particle in various clinical situa- tions.G-emitters should be more effective than F-emitters in the treatment of large solid tumor masses. In large solid tumor masses, the vasculature may be unevenly distributed, and target antigen expression within the tumor may vary.
As a result, the distribution of antibody binding is not necessarily uniform, and
many malignant cells may escape binding by the antibody. Because G-particles have a long range, they should damage malignant cells not directly bound by antibody molecules, resulting in a “crossfire effect.” In contrast, because F- particles have a range of only a few cell diameters, they are likely to miss malig- nant cells not directly bound by the antibody, thus failing to eradicate the tumor.
WhereasG-emitters should be more effective in the treatment of larger solid tumors,F-emitters should be more effective in the treatment of micrometastatic disease and circulating tumor cells like leukemia. In both clinical settings, G- emissions may result in significant damage to normal “bystander” cells (9). In a microdosimetric model using single-cell conditions, one cell-surface decay of theF-emitter211At would result in the same degree of cell killing as about 1000 cell-surface decays of the G-emitter90Y (10). Thus, because of the short range and high energy of F particles, F-particle immunotherapy should provide more efficient and specific killing of tumor cells, particularly in the setting of leuke- mia, microscopic disease, or tumors that spread as thin sheets within compart- ments. Based on these considerations, F-particle therapy has been investigated in a variety of settings, including leukemias, lymphomas, gliomas after surgical resection, neoplastic meningitis, and peritoneal carcinomatosis.
MECHANISMS OF RADIATION-INDUCED CELL DEATH Although the mechanisms by which radiation induces cell death are not com- pletely understood, several processes have been implicated (11,12). Radiation induces single- and double-stranded DNA breaks (13), causes the cleavage of sphingomyelin in cell membranes leading to the formation of ceramide and the subsequent induction of apoptosis (14), and results in the induction of p53 by ATM-dependent phosphorylation, resulting in delays in the G1 phase of the cell cycle (15,16). Death of cells exposed to F-particles occurs only when the par- ticles traverse the nucleus; low doses of F-particle radiation directed at the nucleus are lethal, whereas high doses directed at the cytoplasm have no effect on cell proliferation (17).
Linear energy transfer (LET) and relative biological effectiveness (RBE) are essential radiobiological concepts. LET refers to the number of ionizations caused by that radiation per unit of distance traveled. F-particles cause a large number of ionizations in a relatively short distance, and thus have a high LET. G-particles andL rays, on the other hand, cause a lower number of ionizations over a longer distance, and therefore have a lower LET. For example, the F-particle emitted by astatine-211 (211At) has a mean range in tissue of 70 μm and a LET of 97 keV/
μm, whereas the G-particle emitted by 90Y has a mean range of 3960 μm and a LET of 0.2 keV/μm (7).
The RBE for a type of radiation (e.g., F-particles) refers to the dose of a reference radiation (usually X-rays) that produces the same biological effect as
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the type of radiation in question. Depending on their emission characteristics, the RBE of F-particles for cell sterilization ranges from 3 to 7. The RBE of a type of radiation is a function of the LET of that radiation; RBE is highest at an LET of about 100 keV/μm (18).F-particle radiation has a high RBE because the LET ofF-particles is often close to 100 keV/μm.
The dependency of RBE on LET can be explained by several differences in the type and extent of cellular damage caused by low- and high-LET radiations.
First, high-LET radiation generally causes more irreparable clustered and double- stranded DNA breaks than low-LET radiation (19,20). The maximum rate of double-stranded DNA breaks occurs at LETs of 100–200 keV/μm because at these LETs, the distance between ionizations caused by the radiation approxi- mates the diameter of double-stranded DNA (2 nm) (18). At LETs below 100 keV/μm or above 200 keV/μm, the rates of double-stranded DNA breaks are lower. Second, high-LET radiation causes more severe chromosomal damage, including shattered chromosomes at mitosis and complex chromosomal rear- rangements, than low-LET radiation (11). The highest frequency of chromo- some breaks and complex chromosomal rearrangements occurs with LETs of approx 100–150 keV/μm (11,19). Third, high-LET F-irradiation causes more pronounced G2-phase delays than low-LET L-irradiation (21,22). The mecha- nisms behind these differences in cell-cycle effects have not been fully eluci- dated, but may be related to differences in gene expression induced by the low- and high-LET radiations (23).
Many studies have demonstrated the high potency of F-irradiation. In one of the first, Munro demonstrated that only a few F-particles traversing a nucleus can kill Chinese hamster fibroblasts (17). Bird et al. investigated the cytotoxicity of helium-3 (3He) ions in Chinese hamster V79 cells in different phases of the cell cycle—either at the G1/S transition (induced by hydroxyurea) or in late S phase (24). About four nuclear traversals of 3He were required to kill cells at the G1/
S transition, whereas five to eight were required in late S phase. Other studies using a variety of cell lines irradiated in vitro with F particles (usually from a
238Pu or 239Pu source) also showed that a mean of about two to six F-particle nuclear traversals are required to kill cells (20,25–30). One “outlying” study found that a larger number (10–20) of F-particle traversals were required to kill a cell (31). In this study, mouse embryo fibroblasts from the C3H 10T1/2 cell line were irradiated with 5.6-MeV F-particles (LET 85 keV/μm). One possible ex- planation for the relatively large number of F-particles required to kill these cells is that the cells, when plated in Petri dishes, became flattened, causing the nuclear surface area to became much larger (mean 313 μm2) than that of the cell lines used in the other studies.
SELECTEDF-PARTICLE-EMITTING RADIOISOTOPES Because more than 100 radioisotopes emit F-particles, and most of them decay too quickly to be of therapeutic use, we will confine our discussion of specificF-emitters to those that have therapeutic potential and have been inves- tigated in animal models or humans.
Actinium-225
In its decay to stable 209Bi,225Ac (half-life, 10.0 d) generates francium-221 (221Fr),217At,213Bi, and lead-209 (209Pb) and emits four F particles (Fig. 1).
225Ac can be produced by the natural decay of uranium-233 (233U) (8) or by reactor- or accelerator-based methods (32). In the first method, 233U decays to thorium-229 (229Th), which then decays with a 7340-yr half-life to 225Ra.225Ra (half-life, 14.8 d) emits an F-particle as it decays to 225Ac. Following the collec- tion of 229Th,225Ra and 225Ac are then separated using a series of ion-exchange columns (33). The second method of production of 225Ac, developed by the Institute for Transuranium Elements in Karlsruhe, Germany, involves the neu- tron irradiation of 226Ra by successive n,G capture decay reactions via 227Ac,
228Th to 229Th (32). Other reactor-based methods are under development.
225Ac has both advantages and disadvantages compared with other F-emitters.
Because it emits four F-particles with each decay and has a relatively long half- life,225Ac is more potent than other isotopes that emit just a single F-particle and have shorter half-lives (34). However, after the decay of 225Ac, the daughter isotopes (221Fr,217At,213Bi, and 209Pb) are released from the chelator and can therefore result in nonspecific cytotoxicity. One strategy to overcome this ob- stacle is the use of internalizing antibodies so that the daughters remain predomi- nantly within target cells.
Bismuth-213
213Bi (half-life, 45.6 min) is produced from the decay of 225Ac (Fig. 1). 213Bi decays by a branched pathway to 209Pb and then to stable 209Bi, emitting an F- particle and two G-particles. Additionally, a 440-keV photon emission allows detailed biodistribution, pharmacokinetic, and dosimetry studies to be performed.
A clinically approved 213Bi generator consists of 225Ac dispersed onto a cation exchange resin. The 213Bi is eluted from the generator, and antibody molecules appended with the C-functionalized trans-cyclohexyldiethylenetriamine pentaacetic acid moiety, CHX-A-DTPA, readily chelate the 213Bi radionuclide (35–37). Clinical studies in acute myeloid leukemia, non-Hodgkin’s lymphoma, and melanoma are underway with this isotope.
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Astatine-211
The halogen 211At (half-life, 7.2 h) decays through a branched pathway (Fig.
2). Each branch results in the production of an F-particle and in decay to stable
207Pb. The F-particle produced by the decay of 211At has a mean energy of 6.8 MeV, a mean LET of 97–99 keV/μm, and a range in tissue of 55–80 μm.211At
Fig. 1. The 229Th decay scheme.
is produced by the bombardment of bismuth with F-particles in a cyclotron via the209Bi(F, 2n)211At nuclear reaction (38). After production, 211At can be iso- lated from the cyclotron target using a dry distillation procedure (39). It has been labeled to various types of carrier molecules, including antibodies and antibody fragments (40), tellurium colloid (41,42), the naphthoquinone derivative 2-me- thyl-1,4-naphthoquinol disphosphate (astato-MNDP) (43), methylene blue (44, 45), 2'-deoxyuridine (29), benzylguanidine (28,46), a derivative of the vitamin biotin (47), and bisphosphonates (48).
There are both advantages and disadvantages to the therapeutic use of 211At- labeled molecules. The 7.2-h half-life enables 211At-labeled constructs to be used even when the targeting molecule does not gain immediate access to tumor cells.
Another advantage is that the polonium-211 (211Po) daughter emits K X-rays that permit photon counting of samples and external imaging for biodistribution studies. A disadvantage is that only a few institutions have cyclotrons capable of producing211At. Furthermore, after internalization into cells, 211At is retained less well than other F-emitting radiometals like 212Bi,213Bi, and 212Pb (49).
Bismuth-212
212Bi has a half-life of 60.6 min and emits an F-particle with a mean energy of 7.8 MeV. 212Bi is produced from the decay of 228Th (Fig. 3). A generator that uses224Ra as the parent radionuclide facilitates the production of 212Bi for label- ing to antibodies (50).212Bi decays by a branched pathway to stable 208Pb (Fig.
3). The thallium-208 (208Tl) produced by the decay of 212Bi emits a 26-MeV L- ray along with other medium to high-energy L-particles that require heavy shield- ing to minimize radiation exposure to personnel and have limited the clinical use of212Bi.212Bi-labeled antibodies have been used in cell lines and animal models but not in humans (51–57).
Fig. 2. The 211At decay scheme. EC = electron capture.
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Lead-212
Like212Bi,212Pb is produced from the decay of 228Th and from a 224Ra gen- erator system (Fig. 3) (50).212Pb has a half-life of 10.6 h and emits a G-particle in its decay to 212Bi.212Pb acts as an F-particle emitter only by serving as a source of212Bi. Thus, when administered clinically, 212Pb would serve as an in situ212Bi generator.212Pb has been conjugated to antibodies using various chelators (58–60).
Fig. 3. The 228Th decay scheme.
Radium-223
223Ra (half-life, 11.4 d) can be obtained from uranium mill tailings in large quantities. A generator system has been developed using a 227Ac parent (half- life, 21.8 yr). Like 225Ac,223Ra emits four F-particles over its decay scheme. A clinical phase I study using cationic 223Ra in the treatment of skeletal metastases in patients with prostate and breast cancer demonstrated pain relief and reduction in tumor marker levels (61).
RADIOLABELING
Several techniques can be used to label F-emitting radioisotopes to carrier molecules. For a labeling technique to be useful, it must produce the immun- oconjugate in high yield in a time compatible with the half-life of the radioiso- tope. Furthermore, it must not alter the specificity and affinity of the carrier molecule for the target antigen. Finally, the radioimmunoconjugate must be stable in vivo. Finding a suitable approach for labeling radioisotopes to carrier molecules has often been a limiting factor in the development of radiolabeled molecules for therapeutic use.
Many F-emitters—including 225Ac, 212Bi, and 213Bi—require bifunctional chelators that bind both radioisotopes and carrier proteins. Figure 4 depicts the molecular structures of a number of chelators that have been developed. Many of these chelators are derived from diethylene triamine penta-acetic acid (DTPA), including the isobutylcarboxylcarbonic anhydride derivative (54,55), the cyclic dianhydride derivative (CA-DTPA) (51,56), 2-(p-isothiocyanatobenzyl)-DTPA (SCN-Bz-DTPA) (62), 2-(p-isothiocyanatobenzyl)-5(6)-methyl-DTPA (Mx- DTPA) (62), the cyclohexyl derivative (Cy-DTPA) (63), and the cyclohexylbenzyl derivative (CHX-A-DTPA) (52,64). 1,4,7,10-tetraaza-cyclododecane-1,4,7,10- tetraacetic acid (DOTA) (62,63) and its derivatives (34) have also been used. The isobutylcar-boxylcarbonic anhydride and the cyclic dianhydride of DTPA are not useful in chelating bismuth radioisotopes to antibodies because the radioimmu- noconjugates produced have poor stability in vivo (58,62). Use of SCN-Bz-DTPA, Mx-DTPA, and DOTA results in improved stability of bismuth-labeled antibodies (62). However, the formation of bismuth-DOTA complexes is slow (63). The most effective bismuth chelator to date has been CHX-A-DTPA, which can link bismuth radioisotopes to a variety of antibodies. When CHX-A-DTPA is used to label bismuth to the anti-CD33 antibody HuM195, the labeling procedure takes a mean of 23 min and results in a radiolabeling reaction efficiency of 81% and in production of a final product of 99% purity (64). The resulting immunoconjugates are stable (52,65) and have been used effectively in human clinical trials (36,37). Although the development of a chelator that binds 225Ac has been slow (66), bifunctional derivatives of DOTA were recently found to be effective (34).225Ac-DOTA-con- taining radioimmunoconjugates are stable both in vitro and in mice.
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Fig. 4. Chemical structures of selected chelators derived from diethylene triamine penta- acetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).
211At has been labeled to antibodies and to other types of carrier molecules.
211At is usually labeled to antibodies by incorporation of an aryl carbon–astatine bond into the antibody, not by use of a chelator (38). Various methods can be used to create the aryl carbon–astatine bond, most of which involve an astatodeme- tallation reaction using a tin, silicon, or mercury precursor (38,39). When 211At has been labeled to other carrier molecules besides antibodies, different labeling methods have been used; a complete discussion of these methods is beyond the scope of this review.
DOSIMETRY
The dosimetry of F-emitting radionuclides is distinguished from that of G- emitters by a number of characteristics. Few F-particle emitters decay to stable or short-lived daughter products. Those that do have half-lives that are consid- erably shorter than the commonly used G-emitters. Additionally, the shorter range and higher LET of F-particles compared to G-particles result in an RBE for cell sterilization of 3 to 7.
Radioimmunotherapy with short-lived F-particle emitters such as 213Bi results in markedly different pharmacology than with longer-lived G-emitters. With longer-lived isotopes, pharmacokinetics are determined predominantly by the biological clearance of the antibody. The distribution of the antibody within the first several minutes to hours after administration yields residence times that are negligible in proportion to the overall residence times achieved in target and normal organs. In contrast, for 213Bi, with its 46-min half-life, 20% of the total F emissions occur within the first 15 min after injection, and after 3 h, only 6%
of the total emissions remain.
Conventional medical internal radiation dosimetry (MIRD), based on imag- ing-derived pharmacokinetics, can be performed, assuming that all F and elec- tron emissions arising from the decay of the parent are locally deposited. The absorbed dose is given by the cumulated activity concentration, [Ã], multiplied by the energy emitted per decay as electrons, )e, and the F particles, )F. To determine a radiation dose equivalent (in Sieverts), the F particle contribution to the dose should be adjusted for the RBE: D = [Ã] × ()e + RBE ×)F) (67). All of the F-particle emitters considered for radioimmunotherapy yield radioactive daughters. The fate and biodistribution of these daughters following decay of the parent must be considered in dosimetry estimates. If the daughters remain in the vicinity of the parent, then the parameters )e and )F should include the energy associated with their emissions, weighted by the yield of each daughter. If the half-life of the daughters is long in relation to the rate of diffusion of the daughter, information regarding the redistribution must be incorporated into the dosimetric estimates.
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Given the high energy of F-particles delivered over a short range, however, conventional methodologies that estimate mean absorbed dose over a specific organ volume may not always yield biologically meaningful information. Because of the physical properties of F emissions, targeted cells may receive high ab- sorbed radiation doses, while adjacent cells may receive no radiation at all. There- fore, microdosimetric or stochastic analyses that account for the spatial distribution of various cell types and the distribution of F decays within the organ will be necessary to estimate the absorbed dose to tumor cells and normal tissues more accurately. Because the geometric relationship between the radionuclide and the target cell is not uniform, F-particle hits cannot be assumed to be a Poisson distribution. Several distributions have been modeled, and microdosimetric spec- tra, expressed as specific energy probability densities, have been calculated. Based on this work, methods have been developed to perform basic microdosimetric assessments that account for the probability of the number of hits and the mean specific energy from a single hit (68).
PRECLINICAL AND CLINICAL STUDIES Mouse Models
In one of the earliest studies of F-particle therapy in a mouse model of cancer, mice were inoculated intraperitoneally with EL-4 murine lymphoma cells, which express Thy-1.2, resulting in malignant ascites (51). One day later the mice were treated with intraperitoneal injections of either anti-Thy-1.2 conjugated to 212Bi using the cyclic dianhydride of DTPA or relevant controls. Survival in the treated mice was prolonged compared with controls. However, within 2 h after injection, up to 30% of the injected dose of 212Bi was present in the renal collecting system and bladder. Because free 206Bi is excreted in the urine (69), the presence of 212Bi in the kidneys and bladder suggested dissociation of the 212Bi from the antibody.
In another study, athymic nude mice were inoculated intraperitoneally with LS174T colon cancer cells, which secrete the mucin antigen TAG-72 (57). Seven to 13 d later, the mice were treated with intraperitoneal injections of either 212Bi- labeled anti-TAG-72 or controls. The radiolabeled antibody decreased tumor burdens and may have prolonged survival. However, as in the previous study, much of the injected dose of 212Bi was taken up by the kidney, indicating insta- bility of the radioimmunoconjugate.
To overcome the problem of unstable radioimmunoconjugates, a different chelat- ing agent—CHX-A-DTPA—was used to conjugate 212Bi to the anti-gp70 antibody 103A (52). BALB/c mice were inoculated with the Rauscher leukemia virus, result- ing in erythroleukemia. In this model, infected cells express the viral envelope glycoprotein gp70. Treatment of the mice with 212Bi-103A at 8 to 13 d after inocu-
lation resulted in decreased splenic tumor growth and prolonged median survival.
In contrast to the previous studies, the radioimmunoconjugate was stable.
Hartmann et al. used CHX-A-DTPA to conjugate 212Bi to anti-Tac, which targets CD25 (70). In an experiment designed to simulate the treatment of small- volume disease, athymic nude mice were inoculated subcutaneously with murine plasmacytoma cells engineered to express human CD25. Three days later, prior to the development of overt tumor masses, the mice were treated intravenously with either 212Bi-anti-Tac or controls. Treatment with 212Bi-anti-Tac prolonged the time to tumor occurrence and completely prevented the development of tumors in 30% of mice. However, 212Bi-anti-CD25 did not eradicate established tumors in mice. These results support the hypothesis that F-particle radio-immu- notherapy may be more effective in the treatment of small-volume disease than in the treatment of bulky tumors.
Similar results were found in a mouse model of ovarian cancer (60). Female athymic nude mice were inoculated subcutaneously with SK-OV-3 ovarian can- cer cells. Three days later the mice were treated with the anti-HER2/neu mono- clonal antibody AE1 conjugated to 212Pb. The treatment resulted in improved tumor-free survival compared with controls. However, when the treatment was delayed until larger-volume tumors had developed, no beneficial effects occurred.
Only a few studies have directly compared radioimmunotherapy with F-emit- ters to radioimmunotherapy with G-emitters in animal models. In the first, mice with intraperitoneal growth of murine ovarian cancer were treated with 211At- tellurium colloid or with radiocolloids of dysprosium-165 (165Dy), phosphorus- 32 (32P), or 90Y (42). Although all the treatments prolonged survival compared with controls, 211At-tellurium colloid was the most effective and was often cura- tive. Behr et al. investigated the toxicity and antitumor efficacy of the CO17-1A monovalent Fab' fragment labeled with either 213Bi or 90Y in a human colon cancer xenograft model in nude mice (71). CO17-1A is an antibody directed against a glycoprotein found on normal and malignant gastrointestinal cells. At equitoxic doses, 213Bi-labeled Fab' prevented tumor growth and prolonged sur- vival compared with 90Y-labeled Fab'. The maximum tolerated absorbed doses to blood were similar for the two conjugates. Finally, Andersson et al. compared
211At-labeled MOv18 with the G-emitting 131I-MOv18 (72). MOv18 targets a membrane folate-binding glycoprotein on human ovarian carcinomas. A previ- ous study had demonstrated that 211At-MOv18 has antitumor activity when in- jected intraperitoneally into nude mice with microscopic cancer (73). In the comparison study, 211At-MOv18 prevented the growth of microscopic disease more effectively than 131I-MOv18 did (72). Thus, in the few studies that have directly compared equivalent doses of F-emitters and G-emitters in animal mod-
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els,F-emitters have been more effective in preventing tumor growth and pro- longing survival.
Spheroids
Further evidence that F-particle radioimmunotherapy may be most useful in the treatment of small-volume disease comes from studies of spheroids. Sphe- roids are clusters of malignant cells that serve as models for micrometastatic disease. In one study using relatively small spheroids (200 μm in diameter) composed of CD44-expressing cells, 213Bi-labeled anti-CD44 killed a large pro- portion of the cells (74). In another study, 213Bi labeled to the anti-prostate- specific membrane antigen (PSMA) antibody J591 reduced the volume of spheroids containing cells of the prostate cancer line LNCaP-LN3 (75). The amount of reduction in spheroid volume was inversely proportional to initial spheroid size; 130-μm spheroids responded better than 180-μm spheroids. In another study demonstrating the importance of spheroid size, 212Bi bound to the monoclonal antibody NRLU-10 (which targets a carcinoma antigen) and its Fab fragment killed single human colon adenocarcinoma cells but did not shrink large spheroids (450–1000 μm in diameter) (76).
213Bi-Labeled Anti-CD33 for Myeloid Leukemia
Preclinical biodistribution studies using bismuth-labeled anti-CD33 mono- clonal antibody HuM195, which targets myeloid leukemia cells, were con- ducted in BALB/c mice. These studies showed no loss or uptake of isotope to any normal tissues, including the kidneys, which are know to have an avidity for free bismuth (65). Up to 10 mCi/kg of 213Bi-HuM195 could be injected intra- venously into BALB/c mice without significant toxicity. In vitro studies of
213Bi-labeled HuM195 and the murine anti-prostate membrane-specific antigen (PSMA) antibody J591 demonstrated dose-dependent and specific-activity- dependent killing of cells expressing the relevant antigenic targets (77). In nude mice bearing prostate carcinoma xenografts, treatment with 213Bi-J591 decreased prostate-specific antigen (PSA) levels and prolonged survival com- pared with controls (77).
Based on these data, human clinical trials have been performed at Memorial Sloan-Kettering Cancer Center (MSKCC) in patients with myeloid leukemias, using 213Bi conjugated to HuM195 with CHX-A-DTPA. In a phase I dose- escalation trial, 18 patients with relapsed or refractory acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CMMoL) were treated with 0.28–
1.0 mCi/kg of 213Bi-HuM195 in three to seven fractions over 2–4 d (36). Within 10 min of administration, the 213Bi was taken up in the bone marrow, liver, and spleen, where it remained throughout its half-life. No significant uptake was seen in any other organ. The absorbed dose ratios between marrow, liver, and spleen and the whole body were 1000 times greater with 213Bi-HuM195 than
withG-emitting HuM195 constructs used in similar patients in previous trials (67). Toxicities included myelosuppression in all patients and transient minor liver function abnormalities in 6 patients. Fourteen of 15 evaluable patients (93%) had reductions in circulating blasts after therapy, and 14 of the 18 patients (78%) had decreases in the percentage of bone-marrow blasts. However, no complete remissions occurred. This trial demonstrated that 213Bi-HuM195 has antileukemic activity, but the lack of complete remissions suggested that it remained difficult to target one to two atoms of 213Bi to all leukemia cells in patients with advanced disease.
To test the hypothesis that 213Bi-HuM195 should be more effective in the treatment of cytoreduced disease, we are conducting a phase I/II trial in which patients with advanced AML are treated with cytarabine (200 mg/m2/d for 5 d) followed by 213Bi-HuM195 (0.5–1.25 mCi/kg) (37). To date, 12 patients (median age, 63 yr) have been treated. Nine of 10 evaluable patients had reductions in the percentage of bone-marrow blasts. Two patients, both treated at the 1.0 mCi/kg dose level, achieved complete remissions. A third patient achieved a partial remission (4–9% bone-marrow blasts and normalization of peripheral blood counts) that lasted 4 mo. Myelosuppression was common; however, most patients had severe neutropenia and thrombocytopenia prior to therapy. Although these results are preliminary, sequential administration of cytarabine and 213Bi- HuM195 appears safe and can produce complete remissions in patients with advanced AML.
213Bi-Labeled Antibodies As Conditioning
for Nonmyeloablative Allogeneic Marrow Transplantation Although potentially curative for a number of malignancies, allogeneic mar- row transplantation using myeloablative preparative regimens causes significant toxicity. To decrease the toxicity of the procedure, nonmyeloablative preparative regimens have been developed. Many of these nonmyeloablative preparative regimens use low doses of total-body irradiation (TBI), with or without addi- tional chemotherapeutic agents (78,79). Recently, 213Bi-labeled anti-CD45 was investigated as a preparative regimen in dogs (80). Seven dogs were treated with escalating doses of 213Bi-anti-CD45 (0.1 to 5.9 mCi/kg) without marrow trans- plantation. At higher doses, significant declines in peripheral blood counts occurred; 213Bi doses of 3.7 to 5.9 mCi/kg had similar effects on neutrophil counts similar to those of 200 to 300 cGy of TBI. Subsequently, three dogs were treated with 213Bi-anti-CD45 (3.6 to 8.8 mCi/kg) followed by infusion of marrow from DLA-identical littermates and by immunosuppression with mycophenolate mofetil and cyclosporine. In all dogs, engraftment occurred promptly, and stable mixed hematopoietic chimerism occurred as early as 2–3 wk after transplanta- tion. Subsequent studies revealed that doses of >2 mCi/kg of 213Bi-anti-CD45 were necessary to allow engraftment. Toxicities included transient cytopenias
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and liver function abnormalities. One dog developed intractable ascites; at autopsy, histologic examination showed marked periportal fibrosis, irregular sinusoidal fibrosis, and Kupffer cell aggregates.
In a subsequent study by the same group of investigators, four dogs underwent treatment with 213Bi labeled to an antibody directed at the T-cell receptor (TCR) FG followed by marrow transplantation (81). Because T-cells that express TCRFG are thought to be involved in marrow graft rejection (82), interfering with the function of these T-cells could result in sufficient immunosuppression to allow engraftment of allogeneic marrow. The four dogs received doses of
213Bi-anti-TCRFG ranging from 3.7 to 5.6 mCi/kg, followed by marrow trans- plantation from DLA-identical littermates and by immunosuppression. In all dogs, engraftment occurred rapidly, and stable mixed hematopoietic chimerism occurred as early as 1 wk after transplantation. As with 213Bi-anti-CD45, toxici- ties included transient cytopenias and liver-function abnormalities. One dog developed sustained liver-function test abnormalities; at autopsy, hepatic peri- cellular and periportal fibrosis was found. These studies show that targeted F- particle therapy alone can provide adequate immunosuppression to permit nonmyeloablative marrow transplantation.
225Ac Atomic Nanogenerators
225Ac has been stably conjugated to a variety of antibodies using derivates of DOTA. In vitro studies of 225Ac-J591,225Ac-HuM195, and 225Ac-B4 (anti-CD19) demonstrated dose-dependent and specific-activity-dependent killing of tumor cells at doses 1000 times less than 213Bi-containing constructs (34). In nude mice bearing prostate cancer xenografts, single nanocurie-level doses of 225Ac-J591 decreased PSA levels and cured a substantial fraction of animals (34). Similarly, in mice bearing lymphoma xenografts, treatment with 225Ac-B4 also improved survival compared with controls (34). The increased potency of these 225Ac con- structs compared with 213Bi analogs can be explained by the longer (10-d) half-life of225Ac and by the ability of 225Ac conjugates to act as atomic nanogenerators, emitting four F particles within an individual tumor cell as it decays. A phase I trial of225Ac-HuM195 for advanced myeloid leukemias is planned.
211At-Labeled Anti-Tenascin for Gliomas
A group of investigators at Duke University has extensively studied 211At- 81C6, a chimeric antibody that targets tenascin, and other 211At-labeled constructs (30,83–85). Tenascin is an extracellular matrix glycoprotein that is overexpressed on gliomas relative to normal brain tissue (86).211At-labeled 81C6 and 211At- labeled Mel-14, a chimeric antichondroitin sulfate antibody, are cytotoxic to glioma and melanoma cells, respectively (30). Reduction in cell survival to 37% requires a mean of 1–2 hits to the cell nucleus. 211At-labeled 81C6 was investigated in rats that had been injected intrathecally with human rhabdomyosarcoma cells to cause
neoplastic meningitis (83). Treatment with 211At-labeled 81C6 4–8 d after inocu- lation prolonged survival compared with controls.
Based on these data, a phase I dose-escalation trial of 211At-81C6 was initiated in patients with malignant gliomas (85). Surgical resection of the brain tumors was followed by instillation of up to 10 mCi of 211At-81C6 into the tumor cavity.
At the time of a preliminary report, 12 patients (11 with malignant glioma and one with anaplastic oligodendroglioma) had been treated (85). Pharmacokinetic stud- ies and gamma camera images showed that 99% of the 211At decays occurred within the tumor cavity, indicating high in vivo stability and only low levels of leakage into the circulation. Early results suggest that adjuvant therapy with 211At- 81C6 prolonged survival in these patients, compared with historical controls. No dose-limiting toxicities were observed, and dose escalation continues.
211At-Labeled Methylene Blue for Melanoma
A group in the United Kingdom has investigated the use of methylene blue labeled with 211At in the treatment of melanoma (87–89). Methylene blue is a chemical phenothiazine derivative (3,7-dimethylamino-phenazathionium chlo- ride) that binds to melanin and is taken up by melanoma cells in vitro and in vivo (87). In one study, female nude mice were injected with HX34 human melanoma cells, followed by treatment with 211At-methylene blue either 1 or 7 d later (87).
The treatment decreased the number and size of melanoma metastases. In a second mouse study, 211At-methylene blue inhibited the growth of cutaneous melanomas and spontaneous lymph-node metastases (90). Based on these and other data, human clinical trials were initiated in 1997 (88).
211At- and 213Bi-Labeled Rituximab for Lymphoma
Aurlien et al. studied the effects of 211At labeled to the chimeric anti-CD20 antibody rituximab on CD20-expressing RAEL and K422 lymphoma cells in vitro (91, 92).211At-rituximab bound more strongly to lymphoma cells than to control bone-marrow cells and was cytotoxic to both lymphoma cell lines.
Biodistribution studies in BALB/c mice showed targeting of blood, lung, liver, heart, and other organs. The biodistribution of 211At-rituximab was similar to that of125I-rituximab. The cytotoxicity of 211At-labeled rituximab was not compared with that of 125I-labeled rituximab or unlabeled rituximab. A clinical trial of
211At-rituximab in patients with relapsed B-cell lymphomas is planned.
A phase I clinical trial of 213Bi-labeled rituximab in patients with refractory B-lineage non-Hodgkin’s lymphoma is underway (93). In preclinical studies,
213Bi-rituximab was cytotoxic to lymphoma cells. The construct was stable in vivo and caused no toxic side effects other than myelosuppression. In the clinical trial, nine patients with refractory lymphomas were treated with escalating doses of 213Bi-rituximab (10 to 44 mCi) (93). Grade 1 leukopenia occurred in two patients. One patient achieved a minimal response, and another had stable dis- ease. Enrollment to the study continues.
124 Burke, Scheinberg, and Jurcic
Pretargeting Studies
In an effort to reduce radiation doses to normal organs and improve tumor-to- normal organ dose ratios, pretargeted methods of radioimmunotherapy have been developed. Most of these techniques take advantage of the rapid, high- affinity, and specific binding between streptavidin and the small molecule biotin (94). First, a monoclonal antibody or engineered targeting molecule, conjugated to streptavidin, is administered. Second, a clearing agent, consisting of biotinylated galactosyl human serum albumin, is given after the antibody- streptavidin construct has bound to tumor targets. The biotin component of the clearing agent binds to the streptavidin component of the circulating antibody- streptavidin conjugate. The galactose moiety of the clearing agent then binds to galactose receptors on hepatocytes, and the complex is cleared from the circu- lation. Finally, radiolabeled biotin is administered. Because excess antibody- streptavidin conjugated has been cleared from the circulation, the radiolabeled biotin can bind specifically to “pretargeted” streptavidin at the tumor. Unbound radiolabeled biotin is rapidly excreted in the urine.
Zhang et al. have utilized the pretargeting approach in a murine model of adult T-cell leukemia (ATL) (95). Humanized anti-CD25 was conjugated to strepta- vidin and injected into nonobese diabetic/severe combined immunodeficient mice bearing the ATL cell line MET-1, which expresses CD25. After adminis- tration of the clearing agent, 213Bi-labeled or 90Y-labeled DOTA-biotin was given. Tumor-bearing mice treated with 213Bi had reductions in the concentra- tions of surrogate tumor markers human G2-microglobulin and soluble CD25 as well as improved survival compared with controls. Treatment with 213Bi was more effective than treatment with 90Y. Furthermore, use of the pretargeted approach was more effective than use of 213Bi linked to an intact monoclonal antibody. Despite these exciting and encouraging results, a single course of therapy with 213Bi did not completely eliminate the leukemia from the mice.
POTENTIAL TOXICITIES
In deciding whether to bring an F-emitting radioimmunoconjugate from an animal model into human clinical trials, both efficacy and potential toxicities need to be considered. For example, in one study, 213Bi-labeled anti- thrombomo-dulin targeted pulmonary blood vessels and destroyed pulmonary micrometastases in a mouse model (96). However, the treatment was compli- cated by pulmonary fibrosis. Attempts to avoid pulmonary fibrosis by the use of several techniques to block tumor necrosis factor (TNF)-F—antibodies to TNF-F, the dimeric fusion protein etanercept, and mice genetically deficient for TNF-F production—were unsuccessful (97). Therefore, until the problem of pulmonary fibrosis can be circumvented, the use of 213Bi-labeled anti- thrombomodulin would seem unfit for clinical trials.
Several studies have investigated the toxicity of 211At—either free or bound to carrier molecules. Like iodine (another halogen), 211At is selectively concen- trated in the thyroid gland. Intravenous injections of potentially lethal doses of sodium [211At]astatide in mice resulted in ablation of thyroid follicles as well as reduction of lymphocytes in the blood, spleen, and lymph nodes, marrow toxic- ity, abnormalities of the testes and ovaries, necrosis of the submandibular glands, and necrosis in the crypts of the stomach (98). Lower doses of [211At]astatide produced fibrosis of the thyroid, mild cytopenias, and severe reduction in repro- ductive cells in the testis (99). In a study designed to determine the lethal dose of [211At]astatide in two mouse models, tail-vein injections caused dose-related toxicity to the bone marrow, heart, testes, spleen, and stomach (100).
[211At]astatide may also cause cancer in rats (101). When high doses of 211At were labeled to the antibody 81C6 and administered to mice, perivascular fibro- sis of the intraventricular septum of the heart, myelosuppression, splenic white pulp atrophy, and spermatic maturational delay occurred (102). When labeled to methylene blue and administered to mice bearing melanoma xenografts, 211At caused minor abnormalities in the thyroid gland, regional lymph nodes, and lungs (103,104).
OtherF-emitters have different toxicity profiles. Hepatic toxicity has been observed in several studies of 213Bi-labeled antibodies (36,80,81). Mice treated with lethal doses of 225Ac-HuM195 developed gastrointestinal sloughing and marrow hypoplasia (David Scheinberg, unpublished data). Cynomolgus mon- keys given repeated injections of 225Ac-HuM195 over a 9-month period devel- oped renal failure, anemia, and hepatic injury (David Scheinberg, unpublished data). These toxicities are likely related to the effects of F-emitting daughters produced by the decay of 225Ac. The use of chelators to scavenge these free daughter isotopes from the circulation is a promising approach to ameliorate potential toxicity.
SUMMARY
The role of monoclonal antibodies in the treatment of cancer is increasing.
Most radioimmunotherapy trials have been performed with G-emitting isotopes.
In contrast to G-emitters, the shorter range and higher LET of F-particles allow for more efficient and selective killing of individual tumor cells. Although some experimental models indicate that F-particle immunotherapy may eradicate large tumor burdens, the physical properties of F-irradiation and the clinical trials to date suggest that radioimmunotherapy with F-emitters may be best suited for the treatment of small-volume disease. While results of early studies appear prom- ising, there are several obstacles to the widespread use of F-particle immuno- therapy. To address these difficulties, new sources and methods of production of F-emitters and improved chelation chemistry must be developed. Additional
126 Burke, Scheinberg, and Jurcic
preclinical and clinical investigations are necessary to define optimal radioiso- topes, dosing regimens, and therapeutic strategies.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Christophe Antczak for his expertise in chelation chemistry and assistance in preparing this manuscript.
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