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Basic Science Investigations |
1 Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington
2 Department of Radiation Oncology, University of Washington, Seattle, Washington
3 Department of Medicine, University of Washington, Seattle, Washington
4 NeoRx Corporation, Seattle, Washington
5 Department of Bioengineering, University of Washington, Seattle, Washington
6 Department of Biological Structure, University of Washington, Seattle, Washington
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Key Words: immunoscintigraphy • CD20 • pretargeting • streptavidin • biotin • lymphoma
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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CD20-expressing lymphomas appear to be excellent targets for pretargeted RIT because they are extremely radiosensitive, internalize CD20 antibodies slowly, and have exhibited impressive response rates with chimeric and directly labeled anti-CD20 antibodies (19). Furthermore, temporary eradication of normal B-lymphocytes with malignant lymphoma cells is well tolerated in patients because normal B-lymphocytes killed during therapy are replaced by lymphoid progenitor cells before serious humoral immunodeficiency develops (20).
The suitability of a radionuclide for RIT depends on a variety of factors, such as the nuclides’s radiophysical properties, the target tumor’s morphology and physiology, the antibody’s targeting kinetics, the in vivo stability of the nuclide, the nuclide’s accessibility, and, finally, the availability of simple and efficient clinical-scale radiolabeling methods. 131I and 90Y are ß-particle–emitting radioisotopes that have been most widely used for RIT trials. 131I has been used in the earliest clinical RIT trials because of its ready availability, low cost, simple protein labeling chemistry, the presence of 
-emissions suitable for gamma-camera imaging, and an 8-d half-life (21). 90Y is a pure ß-emitter and emits high-energy, long-range ß-particles that make 90Y suitable for the irradiation of large tumor masses. Moreover, 90Y and similar metallic radionuclides are "residualizing" labels and remain securely sequestered inside lysosomes after endocytosis (21,22).
Various systems have been proposed for the pretargeted delivery of radioactivity to tumors (23). Biotinylated or avidin-conjugated mAbs that bind radiolabeled avidin or biotin, respectively, have been found to be effective methods for the pretargeted delivery of radioactivity to tumors. The avidin-biotin system has many advantages, including the very high affinity of avidin and streptavidin (sAv) for biotin and the multivalency of the system (each sAv or avidin can bind 4 biotin molecules) (19,24). Our laboratory is currently investigating a 2-step pretargeting approach using sAv-conjugated anti-CD20 mAbs in an athymic mouse xenograft system. This report documents the superior tumor localization, imaging, toxicity, and therapeutic efficacy of a pretargeted anti-CD20 antibody compared with the same antibody administered after conventional, direct radiolabeling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Six- to 8-wk-old female BALB/c nude mice (B & K Universal, Kent, WA) were injected subcutaneously with 10 x 106 Ramos cells in each flank. After 7–10 d, palpable tumors appeared and mice with similar tumor sizes (
5-mm diameter) were used for experiments. Tumor-bearing mice were placed on a biotin-free diet (Harlan Teklad, Madison, WI) for 4–7 d before injection of mAb-sAv conjugates and radiobiotin. Animal studies were conducted under the supervision of veterinarians from the University of Washington Comparative Medicine Department or the Animal Health Resources Department of the Fred Hutchinson Cancer Research Center.
Antibodies and sAv Conjugates
The murine anti-human CD20 IgG2a mAb 1F5 was produced in a hollow fiber bioreactor system in the Fred Hutchinson Cancer Research Center Monoclonal Antibody Facility (Seattle, WA). The 1F5 hybridoma was a gift of Dr. Clay Siegall (Seattle Genetics Inc., Seattle, WA). Two irrelevant antibodies, isotype-matched NR-LU-10 (gift from NeoRx Corp., Seattle, WA) and G3G6 (gift from Dr. Dana Matthews, Fred Hutchinson Cancer Research Center, Seattle, WA), were used as nonspecific controls. A conjugate of recombinant sAv and 1F5 was prepared as described (19) using a variation of the method of Hylarides et al. (25). Briefly, sAv was concentrated to 20 mg/mL and reacted with a 3 molar excess of succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC; Pierce, Rockford, IL) for 30 min at pH 8.0. Unreacted SMCC and reaction by-products were removed using G-25 chromatography equilibrated in phosphate-buffered saline (PBS). The 1F5 was concentrated to 10 mg/mL at pH 8.0 and dithiothreitol (Pierce) added to a final concentration of 20 mmol/L. After a 30-min reaction, residual dithiothreitol was removed from the reduced 1F5 using G-25 chromatography as described. After G-25 purification, sAv contained 1.5 reactive maleimides and 1F5 contained 8.4 thiols per molecule as determined using the 5,5'-dithiobis-(2-nitrobenzoic acid) (Pierce) assay described by Hylarides et al. (25).
Sodium chloride was added to the reaction mixture to a final concentration of 0.4 mol/L, glycine was added to 0.05 mol/L, and the pH was adjusted to 9.2 with 1N NaOH. The crude conjugation mixture was applied over an iminobiotin column (Pierce) equilibrated in 0.05 mol/L glycine, 0.5 mol/L NaCl, pH 9.2. Unreacted 1F5 flowed through the column and unreacted sAv and 1F5-sAv conjugate eluted with 0.2 mol/L acetate buffer, pH 4.0. The eluted fraction was diluted by buffer exchange to a conductivity of <2.5 mS/cm with distilled H2O and the pH was adjusted to 6.5 with 1N NaOH. The conjugation mixture obtained from the iminobiotin column was purified by cation-exchange chromatography using a Fractogel EMD SO3- (S) column (EM Separations Technology, Gibbstowin, NJ) equilibrated in 20 mmol/L sodium phosphate buffer, pH 6.5, as described (19). After the load, the column was washed with equilibration buffer to remove the unreacted sAv. The 1F5-sAv conjugate was eluted with 90 mmol/L NaCl in equilibration buffer. The maximal yield of 1F5-sAv after dual-column purification was 35% (mg protein product/mg protein starting materials). The final 1F5-sAv conjugate contained 80%–85% 1:1 1F5-sAv conjugates, 5%–10% 1:2 1F5-sAv conjugates, and 6%–10% molecules with higher ratios of sAv:1F5. Aggregated conjugate eluted at higher concentrations of NaCl. The immunoreactivity of the purified conjugate was determined using a competitive enzyme-linked immunosorbent assy. The biotin binding capacity was determined by displacement of 2-(4'-hydroxyphenylazo)benzoic acid (Aldrich, Milwaukee, WI) from sAv as described (26). The biotin binding capacity of 1F5-sAv was 5 mol of biotin/mol of conjugate. These data confirm high-performance liquid chromatography (HPLC) results showing that some 1F5-sAv conjugates contain 2 or more sAv moieties per antibody molecule.
Clearing Agent
A completely synthetic clearing agent (CA) was used to remove excess unbound mAb-sAv conjugates from the bloodstream before radio-biotin administration. The synthesis and characterization of this reagent have been published elsewhere (27,28). This agent, designated biotin-LC-NM-(Gal-NAc)16 (molecular weight = 8651.6), is a bifunctional moiety consisting of biotin joined, through a modified aminocaproyl spacer, to the core of a 4-generation dendrimeric backbone formed using repetitive bifunctional units (27,28). The outer dendrimeric shell is functionalized with 16 modified N-acetyl-galactosamine residues through aminopentyl linkages. The agent is administered in stoichiometric dose excess to the mAb-sAv conjugate. It has a volume of distribution that allows it to access and bind to vascular and extravascular conjugates. The N-acetyl-galactosamine residues have a high affinity for hepatic asialoglycoprotein receptors, which mediate the rapid hepatic clearance of residual 1F5-sAv conjugates from the bloodstream and their endocytosis into liver cells. Through competitive in vitro binding assays, it has been determined that, although the affinity of this CA for sAv is quite high, it is nonetheless significantly lower than that of natural biotin or of the subsequently administered radionuclide-biotin-chelate ligand (27,28). Thus, although the CA may also bind significantly to extravascular conjugate under conditions of high doses, it does not compromise the binding of subsequently administered biotin-chelate ligand. Numerous studies have been performed documenting that the biotin-chelate ligand can effectively compete with the CA under in vivo conditions and time frames (27,28).
Radiolabeling of mAbs
Radioiodination of mAb-sAv with 131I or 125I (NEN Life Science Products, Inc., Boston, MA) was performed using the chloramine-T method as described (29).
mAbs were radiolabeled with 111In or 90Y (NEN Life Science Products, Inc., Boston, MA) using p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA; Macrocyclics, Richardson, TX) by the method of Mirzadeh et al. (30). Buffer solutions were prepared using metal-free reagents, and storage vials and the mAbs were made metal free using Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA). The radiochemical purity of the conjugates was consistently >95% by thin-layer chromatography.
111In- or 90Y-DOTA-Biotin
The bifunctional ligand DOTA-biotin, a gift from NeoRx Corp. (Seattle, WA), was synthesized as described (31). Carrier-free 111In-chloride (NEN Life Science Products), 0.02–0.5 mL in 0.04 mol/L HCl, was diluted with 0.5 mL of 2 mol/L ammonium acetate, pH 5. DOTA-biotin, 0.1–1 mg, was added, and the solution was heated for 30 min at 80°C. Diethylenetriaminepentaacetic acid (DTPA), 0.05 mL of a 0.1 mol/L solution, was added to chelate any unbound 111In. Carrier-free 90Y-Cl3 (NEN Life Science Products), 0.02–0.2 mL in 0.05 mol/L HCl, was diluted with 2 mol/L ammonium acetate, pH 5, to a total volume of 0.4 mL. Ascorbic acid, 0.05 mL of a 0.5 g/mL solution, and 0.1 mL of 10 mg/mL DOTA-biotin were added, and the solution was heated at 80°C for 1 h. DTPA, 0.05 mL of a 0.1 mol/L solution, was added to chelate any unbound radiometal. The labeling efficiencies for 111In- and 90Y-DOTA-biotin were >94% as assessed using an avidin-bead method and HPLC (19).
Radioimmunoscintigraphy
Nude mice bearing lymphoma xenografts were divided into 2 groups. The pretargeted group was injected intraperitoneally with 1.4 nmol of 1F5-sAv (300 µg) followed 24 h later with 5.8 nmol of CA. Three hours later, the mice were anesthetized with intraperitoneal pentobarbital (50 mg/kg of body weight) and were injected with 1.2 nmol (1 µg) of DOTA-biotin labeled with 11.1 MBq (300 µCi) of 111In in a tail vein and gamma-camera imaging was performed after 0, 2, and 24 h. The control or conventional radioimmunoscintigraphy group of mice were treated by injecting 1.4 nmol (215 µg) of 1F5 directly labeled with 11.1 MBq (300 µCi) of 111In. After the injection of 111In-biotin in pretargeted mice and 111In-mAb in conventional radioimmunoscintigraphy mice, the mice were imaged with a model 400AT gamma camera (General Electric Medical Systems, Milwaukee, WI) equipped with a high-energy collimator at 0-, 2-, and 24-h time points. After 24 h, mice were euthanized, and tumors and normal tissues were excised and assayed for radioactivity.
RIT
To compare the therapeutic efficacies of pretargeted and conventional radiolabeled antibodies, groups of 5 tumor-bearing mice were injected with 1.4 nmol (215 µg) and 7.4 or 14.8 MBq (200 or 400 µCi) of directly labeled 90Y-DOTA-1F5, equimolar amounts (300 µg) of 1F5-sAv or NR-LU-10-sAv conjugates, followed 24 h later by 5.8 nmol (50 µg) of CA and 3 h later by 1.2 nmol (1 µg) of DOTA-biotin labeled with 14.8 or 29.6 MBq (400 or 800 µCi) of 90Y. Mice were monitored every other day for general appearance, tumor volume, and body weight. Mice were euthanized if tumors became large enough (1–1.5 cm in diameter) to cause obvious discomfort or impair ambulation. Differences in mean tumor sizes were analyzed with the Student t test and differences were considered significant at the P < 0.05 level.
Toxicity Analysis
Hematopoietic, renal, and liver toxicity were assessed in BALB/c athymic mice. To assess toxicities beyond 10 d without the confounding effects of euthanasia for rapidly growing tumors, it was necessary to perform these studies in mice that were not bearing tumors. Mice were injected with 1.4 nmol (215 µg) and 7.4 or 14.8 MBq (200 or 400 µCi) of directly labeled 90Y-DOTA-1F5, equimolar amounts (300 µg) of 1F5-sAv conjugates, followed 24 h later by 5.8 nmol (50 µg) of CA and 3 h later by 1.2 nmol (1 µg) of DOTA-biotin labeled with 14.8 or 29.6 MBq (400 or 800 µCi) of 90Y. Blood samples were drawn on days 0, 7, 14, 28, and 35, and the number of leukocytes, neutrophils, and platelets was measured by a CELL-DYN 3500R instrument (model 3500 CS; Abbott Laboratories, Inc., North Chicago, IL). To evaluate hepatotoxicity and nephrotoxicity, serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine (CRE) levels were measured on days 0, 7, 14, and 28 using a Synchron LX 20 instrument (Beckman Coulter, Inc., Fullerton, CA). Duplicate samples were collected from each mouse and the average was reported.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Mice treated with PBS alone or with the control NR-LU-10-sAv conjugate followed by 29.6 MBq (800 µCi) of 90Y-DOTA-biotin experienced no tumor regression and were euthanized between days 13 and 16 because of the exponential growth of their tumor xenografts (Fig. 5A). Mice treated with 7.4 MBq (200 µCi) of conventional 90Y-DOTA-1F5 exhibited a tumor growth delay of 29 d but no complete remissions were observed and all mice eventually required euthanasia as a result of progressive tumor growth (Fig. 5A). Mice treated with 14.8 MBq (400 µCi) of conventional 90Y-DOTA-1F5 experienced a very slow tumor growth but all mice in this group experienced an average 17.3% ± 6.32% loss of their initial body weight and died by day 13 because of toxicity (Fig. 5B).
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Toxicity Analysis
Hematopoietic, hepatic, and renal toxicities were assessed in BALB/c, athymic mice injected with either conventional or pretargeted RIT protocols. To assess toxicities beyond 10 d without the confounding effects of euthanasia for rapidly growing tumors, it was necessary to perform these studies in mice that did not bear tumors. As shown in Figure 6A, mice injected with 7.4 MBq (200 µCi) of conventional 90Y-DOTA-1F5 exhibited a significant decrease (79.2% ± 0.73%) in the neutrophil count by day 7, which persisted for a week, but the neutrophil count subsequently rebounded to the initial normal value by day 28. Mice injected with 14.8 MBq (400 µCi) of conventional 90Y-DOTA-1F5 exhibited an even more profound decrement in the neutrophil count (87.7% ± 0.69%) by day 7, and eventually all mice in this group died between days 10 and 12 of presumed infections. Neutropenia evolved more slowly and was less marked in the pretargeted groups injected with either 14.8 or 29.6 MBq (400 or 800 µCi) of 90Y-DOTA-biotin (Fig. 6A). The neutrophil nadir occurred on day 14 in both pretargeted groups, with a mean nadir neutrophil count of 0.63 ± 0.32 x 103/mm3 in the 14.8-MBq (400 µCi) group and 0.31 ± 0.27 x 103/mm3 in the 29.6-MBq (800 µCi) group. In the pretargeted group injected with 29.6 MBq (800 µCi) of 90Y-DOTA-biotin, a mean decrease of 85.3% ± 0.53% in the neutrophil count was observed, similar to the decrement observed with 7.4 MBq (200 µCi) of conventional 90Y-DOTA-1F5. In both pretargeted groups, the original normal neutrophil counts were regained by day 35. A similar pattern was observed with the total leukocyte counts (Fig. 6B) and platelet counts (Fig. 6C). Thrombocytopenia was significantly less marked with 29.6 MBq (800 µCi) of pretargeted 90Y-DOTA-biotin than with 7.4 MBq (200 µCi) of conventionally targeted 90Y-1F5 (7.79 ± 0.66 x 105/mm3). No significant changes in the hematocrit (Fig. 6D) were observed in either the 14.8- or 29.6-MBq (400 or 800 µCi) pretargeted group. However, in the conventional RIT group given 7.4 MBq (200 µCi) of 90Y-DOTA-1F5, a marked reduction in the hematocrit was observed with a nadir hematocrit of 25.4% ± 1.8% (46.4% decrease). Anemia resolved by day 35. The hematocrit fell significantly (15.2%) by day 7 in the 14.8-MBq (400 µCi) conventional RIT group, but complete assessment was not possible because all mice died of hematopoietic toxicity by day 12.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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The markedly superior biodistributions of radioactivity using the anti-CD20 pretargeting approach demonstrated by radioimmunoscintigraphy and by -counting of excised organs suggest that pretargeting would also afford superior therapeutic efficacy and diminished toxicity compared with conventional RIT. These predictions have been confirmed by the RIT experiments presented in this article and in previous studies (17,19). Conventional 90Y-1F5 antibodies were unable to cure animals bearing Ramos lymphoma xenografts at sublethal radiation doses of <14.8 MBq (<400 µCi). In marked contrast, tumor-bearing mice were reliably cured using pretargeted anti-CD20 RIT at optimal doses. Importantly, the curative doses of 1F5-sAv + 90Y-DOTA-biotin used were well tolerated by mice.
Various RIT studies have clearly shown that directly labeled mAbs generally induce marked levels of hematotoxicity because of the presence of high levels of radioactivity in the bloodstream for prolonged periods of time (32,33). Our study demonstrates that this hematotoxicity can be reduced significantly using pretargeting, which is capable of delivering higher amounts of radioactivity to tumors without increasing marrow exposure to radioactivity. In this study, hematologic toxicity was significantly lower with pretargeted RIT, even after the administration of 29.6 MBq (800 µCi) of 90Y, which was at least twice a 100% lethal dose (14.8 MBq or 400 µCi of 90Y) of the directly conjugated antibody. In contrast, the prolonged serum half-life of directly labeled 1F5 led to substantial toxicity at 7.4 MBq (200 µCi) and fatal toxicities at 14.8 MBq (400 µCi) of 90Y. Detailed serial analyses of hematologic parameters confirmed that leukopenia, neutropenia, anemia, and thrombocytopenia were all significantly reduced in mice treated with the pretargeting approach compared with conventional RIT. The reduced toxicity of pretargeted RIT enabled the safe delivery of radioactivity at twice the lethal dose of directly labeled antibodies and resulted in 90% apparent cures. The CA appears to play a key role in the removal of excess 1F5-sAv immunoconjugate from circulation and, thereby, improves the tumor-to-normal organ ratios, leading to the enhancement of the therapeutic index.
Significant nephrotoxicity and hepatotoxicity were not observed in these experiments in either the pretargeted or the conventional RIT group. However, delayed hepatotoxicity, nephrotoxicity, and radiation cystitis might occur with longer follow-up as a result of the urinary excretion of large doses of 90Y-DOTA-biotin. To assess this possibility we are conducting long-term experiments with mice treated with pretargeted RIT and large doses of 90Y-DOTA-biotin up to 44.4 MBq (1,200 µCi) with serial measurements of liver transaminases and serum CRE over a period of 1 y. At the end of the year, mice will be euthanized and the liver, bladder, and kidneys will be examined histologically for evidence of radiation injury or fibrosis.
The promising results of our experiments using murine xenograft models suggest that clinical trials using pretargeted RIT might permit substantial dose escalation of CD20-directed RIT to radiation doses that would produce higher rates of complete remission and improved cure rates in lymphoma patients with tolerable toxicity and without the need for stem cell rescue. Despite the fact that pretargeting appears very promising, this method also has limitations. Among the disadvantages of pretargeting are the requirement for separate, sequential injections at defined time intervals and the immunogenicity of sAv (34). Other reservations can also be identified that mandate caution in extrapolating these murine experiments to humans. First, radioimmunoconjugates may have more uniform tumor uptake in this mouse xenograft system than that in humans because of the improved vascularity of the tumor xenografts. Second, mouse B-lymphocytes do not bind anti-CD20 antibodies but human B-lymphocytes will, which might adversely impact targeting in patients unless circulating B-cells are precleared by injection of nonradioactive anti-CD20 antibody before RIT. Third, the relatively long pathlength of ß-emissions of 90Y in relation to the size of the mouse and the size of the tumor xenografts may be considered disadvantageous. On the other hand, if one considers the enhanced toxicity to a greater volume of normal tissue in the mouse model from ß-particles emanating from the tumors that deposit their energy outside the xenograft, mouse xenograft studies using 90Y-immunoconjugates can be conservative predictors of human results (35). Despite these caveats, we believe that this investigation, and those of other investigators (17,19,35), has firmly established that pretargeted RIT can provide remarkable discrimination between tumor and normal tissue and that this enhanced targeting translates to superior RIT efficacy.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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For correspondence or reprints contact: Oliver W. Press, MD, PhD, Fred Hutchinson Cancer Research Center, D3-190, 1100 Fairview Ave. N., Seattle, WA 98109.
E-mail: press{at}u.washington.edu
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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) or 11.6 nmol of CA (
). Blood samples were collected at 6, 22, 25, 27, 29, and 44 h after 125I-1F5-sAv injection and assayed in gamma counter. Data represent mean percentage injected dose per gram (%ID/g) ± SD.
). Data in A were obtained 24 h after injection of radioactivity and are shown as %ID/g ± SD. Data represent the mean of 3 mice in each group.
), with 7.4 MBq (200 µCi) (
) of 90Y-DOTA-biotin. Data represent mean percentage ± SD of initial values (n = 5).