Bioconjugate Chem. 2006, 17, 485−492
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Radiolanthanide-Labeled Monoclonal Antibody CC49 for Radioimmunotherapy of Cancer: Biological Comparison of DOTA Conjugates and 149Pm, 166Ho, and 177Lu Huma Mohsin,† Fang Jia,‡ Geethapriya Sivaguru,‡ Michael J. Hudson,§ Tiffani D. Shelton,| Timothy J. Hoffman,| Cathy S. Cutler,⊥ Alan R. Ketring,⊥ Phillip S. Athey,3 Jaime Simo´n,3 R. Keith Frank,3 Silvia S. Jurisson,†,⊥,# and Michael R. Lewis*,‡,|,#,X Department of Chemistry, and Department of Veterinary Medicine and Surgery, University of MissourisColumbia, Columbia, Missouri 65211, Department of Biology, University of Maryland Baltimore County, Baltimore, Maryland 21250, Research Service, Harry S. Truman Memorial Veterans’ Hospital, Columbia, Missouri 65201, University of Missouri Research Reactor, University of MissourisColumbia, Columbia, Missouri 65211, Dowpharma, The Dow Chemical Company, Freeport, Texas 77541, and Nuclear Science and Engineering Institute, and Department of Radiology, University of MissourisColumbia, Columbia, Missouri 65211. Received August 1, 2005; Revised Manuscript Received February 1, 2006
The radiolanthanides 149Pm, 166Ho, and 177Lu have decay characteristics suitable for radioimmunotherapy (RIT) of cancer. N-Hydroxysulfosuccinimidyl DOTA (DOTA-OSSu) and methoxy-DOTA (MeO-DOTA) were conjugated to the anti-TAG-72 monoclonal antibody CC49 for radiolabeling with 149Pm, 166Ho, and 177Lu. While both DOTA conjugates could be labeled to high specific activity with 177Lu, MeO-DOTA afforded superior conjugate stability, radiolabeling, and radiochemical purity. Pilot biodistributions in nude mice bearing LS174T human colon carcinoma xenografts demonstrated that MeO-DOTA afforded higher tumor uptake and lower kidney retention of 177Lu than DOTA-OSSu. The in vitro stability of 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49 was evaluated using serum and hydroxyapatite assays. Serum stability of radiolanthanide-labeled MeO-DOTA-CC49 followed a trend based on the coordination energies of the radiometals, with 177Lu showing the highest stability after 96 to 168 h at 37 °C. In contrast, MeO-DOTA-CC49 labeled with all three radiolanthanides was >92% stable to hydroxyapatite challenge for 168 h at 37 °C. Comprehensive biodistributions of 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49 were obtained in LS174T-bearing nude mice. Maximum tumor uptakes were 100.0% ID/g for 149Pm at 96 h, 69.5% ID/g for 166Ho at 96 h, and 132.4% ID/g for 177Lu at 168 h. Normal organ uptakes were generally low, except in the liver, spleen, and kidney at early time points. By 96 to 168 h postinjection, nontarget organ uptake decreased to approximately 7% ID/g (kidney), 12% ID/g (spleen), and 20% ID/g (liver) for each radiolanthanide. When labeled with 149Pm, 166Ho, and 177Lu, MeO-DOTA-CC49 has potential for RIT of colorectal cancer and other carcinomas.
INTRODUCTION Radiolabeled monoclonal antibodies (mAbs) have shown great promise for cancer therapy. Recently, two commercial preparations of radiolabeled mAbs have been approved by the United States Food and Drug Administration for radioimmunotherapy (RIT)1 of relapsed or refractory B-cell nonHodgkin’s lymphoma (1, 2). Targeted radiopharmaceuticals are well suited to the therapy of this disease, as tumors are well vascularized for the delivery of the mAbs, and lymphocytes are exquisitely sensitive to radiation injury. However, targeting of solid tumors with therapeutic radiopharmaceuticals has been far * To whom correspondence should be addressed: Michael R. Lewis, Ph.D., Department of Veterinary Medicine and Surgery, College of Veterinary Medicine, 379 E. Campus Dr., University of Missouris Columbia, Columbia, MO 65211. Phone: (573) 814-6000, ext. 3703. Fax: (573) 814-6551. E-mail:
[email protected]. † Department of Chemistry, University of MissourisColumbia. ‡ Department of Veterinary Medicine and Surgery, University of MissourisColumbia. § University of Maryland Baltimore County. | Harry S. Truman Memorial Veterans’ Hospital. ⊥ University of Missouri Research Reactor, University of Missouris Columbia. 3 The Dow Chemical Company. # Nuclear Science and Engineering Institute, University of Missouris Columbia. X Department of Radiology, University of MissourisColumbia.
less effective in producing lasting remissions or tumor control. The slow plasma clearance properties of radiolabeled mAbs result in prolonged exposure of the bone marrow to ionizing radiation. This requires limiting the dose to the tumor to keep marrow exposure below 150 to 200 rad (1.5 to 2.0 Gy). Creation of stable fragments of mAbs has improved clearance kinetics but yielded decreased tumor antigen binding affinity and altered biodistributions, often with unacceptably high kidney doses (3). An alternative strategy is to label mAbs with radionuclides having emission characteristics that are more effective for solid tumor therapy. The radiolanthanides 149Pm, 166Ho, and 177Lu (Table 1) possess a range of half-lives and β- energies for RIT applications. They also emit low abundance γ rays with energies suitable for tracking radiolabeled mAbs in vivo and estimating radiation dosimetry. Furthermore, these radiolanthanides can be 1 Abbreviations: % ID/g, percent injected dose per gram of tissue; % ID/organ, percent injected dose per organ; AUC, area under the curve; BSA, bovine serum albumin; CC49, second generation antiTAG-72 mAb; DOTA, 1,4,7,10-tetraazacyclododecane-N,N′,N′′,N′′′tetraacetic acid; DOTA-CC49, CC49 conjugated with DOTA-OSSu; DOTA-OSSu, N-hydroxysulfosuccinimidyl DOTA; DTPA, diethylenetriaminepentaacetic acid; GF-HPLC, gel filtration HPLC; HA, hydroxyapatite; MeO-DOTA, methoxy-DOTA; MeO-DOTA-CC49, CC49 conjugated with MeO-DOTA; PA-DOTA, 1,4,7,10-tetraaza-N(1-carboxy-3-(4-aminophenyl)propyl)-N′,N′′,N′′′-tris(acetic acid)cyclododecane; RIC, radioimmunoconjugate; RIT, radioimmunotherapy; TAG-72, tumor-associated glycoprotein-72.
10.1021/bc0502356 CCC: $33.50 © 2006 American Chemical Society Published on Web 02/23/2006
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Table 1. Decay Characteristics and DOTA log KML Values for 149Pm, 166Ho, and 177Lu T1/2 (h)
β- MeV (%)
γ keV (%)
log KML (M3+-DOTA)
149Pm
53.1
286 (3.1)
22.87
166Ho
26.9
81 (5.4)
24.83
177Lu
159.6
0.784 (9) 1.06 (90) 1.76 (47) 1.84 (52) 0.497 (90)
208 (11)
25.34
radiometal
obtained readily from a nuclear reactor, by both direct neutron capture and indirect methods (4). Bifunctional derivatives of 1,4,7,10-tetraazacyclododecaneN,N′,N′′,N′′′-tetraacetic acid (DOTA) have been used often to label mAbs with lanthanide radionuclides and other radiometals. We previously developed a facile, water-soluble method (5, 6) for conjugating mAbs with N-hydroxysulfosuccinimidyl DOTA (DOTA-OSSu). Conjugates of several antibodies prepared with DOTA-OSSu have been labeled to high specific activity, high radiochemical purity, and high stability with 111In and 90Y (57), as well as 64Cu (8-10). However, modified structures of DOTA, such as methoxy-DOTA (MeO-DOTA, Figure 1), may have improved binding kinetics and stability for radiolanthanides. Methoxy-DOTA has four amine and four carboxylic acid coordination sites available to bind the radionuclide, while the benzyl isothiocyanate moiety is available off one of the carboxylate arms for conjugation. CC49 (11) is a second generation murine IgG1 mAb that has been shown to react with the disaccharide sialyl Tn epitope on the extracellular antigen tumor-associated glycoprotein-72, or TAG-72. TAG-72 is expressed on ∼85% of human adenocarcinomas such as colon, breast, pancreatic, ovarian, prostate, nonsmall cell lung, and gastric cancers (12-21). Both 131I- and 177Lu-labeled CC49 have been evaluated for RIT in tumorbearing mice (22, 23) and patients (24-29). These studies demonstrated excellent tumor targeting and elimination of established tumors in rodent models. The objective of the present studies was to obtain comparative biodistributions of CC49 labeled with 149Pm, 166Ho, and 177Lu in nude mice bearing LS174T human colon carcinoma xenografts. CC49 conjugates were prepared using DOTA-OSSu and MeO-DOTA, and the stabilities and tumor-targeting properties of the radioimmunoconjugates were evaluated in vitro and in vivo. When labeled with 177Lu, MeO-DOTA was superior to DOTA in terms of CC49 conjugate stability, radiolabeling, radiochemical purity, and biodistributions. The favorable and comparable biodistributions and tumor targeting properties exhibited by the three radiolanthanides allowed the current work to be extended to preclinical RIT studies with each radionuclide.
EXPERIMENTAL PROCEDURES General. The radiolanthanides were obtained as 149PmCl3, 177LuCl in 0.05 M HCl from the University of 3, and 3 Missouri Research Reactor (MURR, Columbia, MO). DOTA was purchased from Macrocyclics (Dallas, TX), and MeODOTA was provided by the Dow Chemical Company (Dowpharma, Freeport, TX). Chelex 100 (Analytical Grade, 100200 mesh, sodium form) was purchased from Bio-Rad (Hercules, CA). All solutions were prepared using ultrapure water (18 MΩcm resistivity). CC49 was produced from the hybridoma cell line provided by Dr. Jeffrey Schlom at the National Cancer Institute (Bethesda, MD). Hybridoma cells were grown in a CELLine CL 1000 bioreactor (Integra Biosciences AG, Chur, Switzerland) in serum-free medium (GIBCO Hybridoma-SFM, Invitrogen, Carlsbad, CA) with ORIGEN (Hybridoma Cloning Factor, IGEN International, Inc., Gaithersburg, MD) for 38 days, and
166HoCl
Figure 1. Structures of chelator-mAb conjugates DOTA-CC49 and MeO-DOTA-CC49.
supernatant from the cell side was harvested every 3-4 days. CC49 was purified from the supernatant by Protein G affinity chromatography, using a 10-mL UltraLink Immobilized Protein G column (Pierce, Rockford, IL). The column was washed with 0.1 M sodium acetate, pH 6.0, and eluted with 0.1 M glycine, pH 3.0, after which collected fractions were neutralized with 1 M Tris, pH 8.0. The pooled fractions containing purified mAb were dialyzed against of 1 L of 10 mM NaH2PO4/150 mM NaCl, pH 7.5, containing approximately 1 g of Chelex 100, for 72 h at 4 °C, with two buffer changes. This procedure afforded typical yields of 85 to 100 mg of CC49. CC49 concentration was measured by absorbance at 280 nm ( ) 1.5 × 105 M-1 cm-1; A280 at 1 mg/mL ) 1.4). The purity of the radioimmunoconjugates was confirmed by gel filtration HPLC (GF-HPLC), using a Waters (Milford, MA) Delta 600 chromatograph equipped with a manual Rheodyne injector, a 2487 dual wavelength UV detector, a Packard 500TR Flow Scintillation Analyzer with a LQ flow cell for 149Pm, 166Ho, and 177Lu, a Waters busSAT/IN analog-digital interface, and the Waters Millennium 32 software package. A Superose 12 HR 10/30 (Amersham Pharmacia, Torrance, CA) column (300 × 10 mm), an isocratic mobile phase of 100 mM NaH2PO4/0.05% NaN3, pH 6.8, and a flow rate of 0.5 mL/min were used. Scintillation counting was performed on a Wallac 1480 Wizard 3′′ automated gamma counter (PerkinElmer Life Sciences, Gaithersburg, MD). Antibody Conjugation. An aliquot of 6.03 mg of CC49 was conjugated with the bifunctional chelating agent N-hydroxysulfosuccinimidyl DOTA (DOTA-OSSu) at a 20:1 molar ratio of DOTA-OSSu:mAb, as described previously (5). For methoxyDOTA (MeO-DOTA) conjugation, a modification of the method of Sumerdon et al. (30) was used. Briefly, an aliquot of 6.03 mg of CC49 in 1 mL of 10 mM NaH2PO4/150 mM NaCl, pH 7.4, was dialyzed against 1 L of 0.1 M Na2HPO4, pH 7.5, containing approximately 1 g of Chelex 100, for 18 to 24 h at 4 °C, and then against 1 L of 0.1 M NaHCO3/Na2HPO4, pH 8.5, containing approximately 1 g of Chelex 100, for 48 h at 4 °C, with one buffer change. CC49 was conjugated with MeODOTA at a MeO-DOTA:mAb molar ratio of 20:1. The reaction mixture was incubated for 3 h at 37 °C and then dialyzed against 0.25 M ammonium acetate, pH 7.0, containing approximately 1 g of Chelex 100, at 4 °C for 144 h, with five buffer changes. The DOTA-CC49 and MeO-DOTA-CC49 conjugate concentrations were measured by absorbance at 280 nm. The average number of chelates per antibody was determined using an 111In isotopic dilution assay method, using a previously published procedure (5). Radiolanthanide Labeling. Modifications of the methods previously described by Lewis et al. (5, 31) were used to radiolabel DOTA-CC49 and MeO-DOTA-CC49. An initial radiolabeling ratio of 5 µCi/µg was used. In the 177Lu labeling reaction, 59.6 µL of 1.0 M ammonium acetate, pH 4.5, was added to 2.0 mCi of 177LuCl3 in 2.0 µL of 0.05 M HCl, followed by 400 µg of DOTA-CC49 or MeO-DOTA-CC49 in 65.6 µL
149Pm-, 166Ho-,
and
177Lu-Labeled
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mAb CC49
of 0.25 M ammonium acetate, pH 7.0. The reaction mixture was incubated at 43 °C for 1 h, and diethylenetriaminepentaacetic acid (DTPA), pH 6.0, was added to a final concentration of 1 mM. The reaction mixture was allowed to stand for 15 min at room temperature and then purified using a Bio-Spin 6 column (Bio-Rad) equilibrated with 10 mM Na2HPO4/150 mM NaCl, pH 7.5. Prior to purification, radiometal labeling efficiency and specific activity were determined by GF-HPLC. After spin column purification, the specific activity of the mAb was adjusted to 1 µCi/µg, by dilution with unmodified CC49. Radiochemical purity was then determined by GF-HPLC. For 166Ho and 149Pm labeling, similar conditions were used as for 177Lu, except the 149Pm solution contained 2.0 mCi in 6.0 µL of 0.05 M HCl. Immunoreactivity Determination. A modification of a previously described method (5) was used. Briefly, a dilution of the purified radiolabeled CC49 conjugate, containing approximately 200 000 cpm of radioactivity, was mixed with a 20-fold molar excess of bovine submaxillary mucin type I-S (Sigma; St. Louis, MO) in 150 µL of 1% human serum albumin/ 10 mM NaH2PO4/150 mM NaCl, pH 7.4. A control sample was prepared in an identical manner, except that mucin type I-S was not added. The reaction mixtures were incubated at 37 °C for 15 min with continuous end-over-end mixing, after which aliquots of 100 µL were analyzed by GF-HPLC, as described above. Immunoreactivity was calculated as the percentage of the total radioactivity shifted to complexes with apparent molecular weights higher than that of the mAb. Serum Stability Studies. An aliquot of 1 mCi of 177LuDOTA-CC49 or 177Lu-, 166Ho-, or 149Pm-MeO-DOTA-CC49 was added to 1 mL of mouse serum, clarified by centrifugation at 16 110g for 15 min and stabilized with 10 µL of 10% NaN3. Serum samples were analyzed by GF-HPLC, as described above, after incubation at 37 °C for 1, 4, 24, 48, 96, and 168 h. Hydroxyapatite Stability Studies. Hydroxyapatite stability studies were performed according to a previously published procedure (32). Reaction mixtures containing 300 µL of HAUltrogel (Sigma, 40% w/w aqueous ethanol suspension, particle size 60-180 µm), 100 µL of bovine serum albumin (BSA) to give a final concentration of 2.5%, 800 µL of 50 mM Tris with 0.01 M CaCl2, and 10 to 20 µCi of radiolabeled MeO-DOTACC49 were incubated in 1.5-mL conical polypropylene tubes with continuous mixing at 37 °C. Each day for 7 days, one reaction mixture was separated into liquid and solid phases. The tubes were centrifuged in an Eppendorf 5415D centrifuge (Brinkmann Instruments, Westbury, NY) at 10 000 rpm for 1 min. The liquid phase was removed. The solid sample was washed with 1 mL of water and recentrifuged at 10 000 rpm for 1 min. The liquid wash was removed from the tube. The solid phase was dissolved in 1.3 N HNO3. The liquid phase, wash, and solid phase were counted in a gamma counter. The percentage of radioactivity in the liquid phase of the sample was calculated by combining the liquid phase and wash and dividing by the total counts in the three parts (liquid, wash, and solid). The solid phase was calculated by taking the counts in the solid phase and dividing by the total counts. Biodistribution Studies. All experiments were conducted in compliance with the guidelines established by the Animal Care and Use Committee of the University of MissourisColumbia Animal Care Quality Assurance Office. Outbred female nu/nu mice (4 to 6 weeks of age) were implanted subcutaneously (sc) in the hind flank with 2 × 106 LS174T human colon cancer cells in 0.15 mL of Hank’s Balanced Salt Solution. After 14 days, tumors had grown to 100-400 mg, with a mean weight of 175 mg. Mice were then injected intravenously (iv) via the tail vein with 60 µCi/60 µg of 177Lu-DOTA-CC49 or 177Lu-, 166Ho-, or 149Pm-MeO-DOTA-CC49.
Table 2. Serum Stability (mean ( SD)a of Radiolanthanide-Labeled MeO-DOTA-CC49 and 177Lu-DOTA-CC49 MeO-DOTAb
DOTAb
time (h)
149Pm
166Ho
177Lu
177Lu
0 1 4 24 48 96 168
99.2 ( 1.3 99.0 ( 1.8 98.1 ( 3.2 97.4 ( 3.7 96.7 ( 4.0 93.7 ( 2.5 91.6 ( 0.5
98.7 ( 0.9 98.6 ( 1.1 98.1 ( 1.4 98.4 ( 1.4 97.7 ( 1.4 93.9 ( 0.7 n.d.c
99.2 ( 1.2 99.6 ( 0.7 100.0 ( 0.0 98.1 ( 2.1 99.3 ( 1.2 96.5 ( 0.9 98.0 ( 2.4
98.3 ( 2.9 98.3 ( 2.9 98.2 ( 3.1 96.7 ( 3.2 97.2 ( 3.6 97.3 ( 4.0 94.3 ( 3.0
a The standard deviations (SD) represent variances of three separate experiments. b n ) 3. c n.d. ) not determined.
For 149Pm- and 177Lu-labeled CC49, biodistributions were obtained at 15 min, 1, 4, 24, 48, 96, and 168 h postinjection. For 166Ho-MeO-DOTA-CC49, biodistributions were obtained at the same time points out to 96 h postinjection. Tissues harvested included blood, lung, liver, spleen, kidney, muscle, fat, heart, bone, uterus, ovaries, bladder, stomach, small intestine, large intestine, tumor, and carcass. Tissues were drained of blood, weighed, and counted in the gamma counter with a standard of the injected dose, such that decay-corrected uptakes were calculated as the percent injected dose per gram of tissue (% ID/g) and the percent injected dose per organ (% ID/organ). Statistical Analysis. To compare biodistributions between 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49, one-way analysis of variance (ANOVA) using statistical software SPSS 12.0.1 (Chicago, IL) was performed. Differences at the 95% confidence level (p < 0.05) were considered significant.
RESULTS Conjugation of DOTA and MeO-DOTA to CC49 and Radiolanthanide Labeling. The reactions of N-hydroxysulfosuccinimidyl-DOTA (DOTA-OSSu) and methoxy-DOTA (MeODOTA) with mAb CC49 are expected to produce amide and thiourea linkages, respectively, between chelators and the -amino group of lysine residues and/or the N-termini of polypeptide chains (Figure 1). Both bifunctional chelating agents were reacted with CC49 at a molar ratio of 20:1. The average number of chelates per antibody was 5.53 for DOTA-CC49, as determined by an 111In isotopic dilution assay (5, 33). In comparison, the average number of chelates per antibody for MeO-DOTA-CC49 was 2.51. Reaction with purified antigen and GF-HPLC analysis demonstrated that the immunoreactivity of the DOTA and MeO-DOTA conjugates of CC49 were 97%. The nonreactive fraction (3%) consisted of incompletely assembled mAb species, which were minor byproducts of hybridoma production. The average labeling efficiency of DOTA-CC49 with 177Lu was 70.8% at a ratio of 5 µCi/µg, and purification by gel filtration spin column chromatography afforded a radiochemical purity of 95% as determined by GF-HPLC. At a 5 µCi/µg ratio, the MeO-DOTA-CC49 conjugate gave average labeling yields of 76.8%, 63.5%, and 68.0%, respectively, with 149Pm, 166Ho, and 177Lu. For each radiolanthanide, radiochemical purity of MeO-DOTA-CC49 was >98.0% after purification, as determined by GF-HPLC. Surprisingly, the radiolabeling efficiency of DOTA-CC49 dropped precipitously and reproducibly to near zero values within two months of storage at either 4 °C or -70 °C. In contrast, after more nine months at 4 °C the MeO-DOTA conjugate could be labeled with all three radiolanthanides with efficiencies up to 80% at 5 µCi/µg. In Vitro Stability Studies. The stability of 177Lu-DOTACC49 in mouse serum was 94.3 ( 4.0% after 168 h at 37 °C (Table 2), a value significantly lower (p ) 0.0067) than that of
488 Bioconjugate Chem., Vol. 17, No. 2, 2006
Figure 2. Percentages of 149Pm, 166Ho, and 177Lu bound to MeODOTA-CC49 as a function of time of incubation with hydroxyapatite at 37 °C. Error bars represent standard deviations (SD) of three separate experiments.
Figure 3. Comparison of 177Lu-DOTA-CC49 and 177Lu-MeODOTA-CC49 biodistributions (n ) 5) in blood, liver, kidney, bone, and LS174T tumor at 24 h postinjection. Error bars represent standard deviations (SD).
(98.0 ( 2.4%). In the case of the MeO-DOTA conjugate (Table 2), the serum stabilities at 96 h were statistically identical for 149Pm (93.7 ( 2.5%) and 166Ho (93.9 ( 0.7%), both lower than the corresponding value for 177Lu (96.5 ( 0.9%). The 177Lu-labeled MeO-DOTA conjugate was more stable than 149Pm-MeO-DOTA-CC49 at 168 h (p ) 0.02) and more stable than 166Ho-MeO-DOTA-CC49 at 48 h (p ) 0.02) and 96 h (p ) 0.03). The radiolanthanides lost from DOTA-CC49 and MeO-DOTA-CC49 were invariably observed as low molecular weight species. Hydroxyapatite stability studies were also conducted, based on a previously published in vitro method for predicting the in vivo stability of radiolanthanide chelates (32). In the hydroxyapatite studies (Figure 2), the MeO-DOTA conjugate was 92.3 ( 2.6% stable with 166Ho, 92.9 ( 2.8% stable with 149Pm, and 95.2 ( 1.0% stable with 177Lu for 168 h at 37 °C. The only statistically significant difference observed was between 177Lu and 166Ho at 168 h, which was marginal at p ) 0.048. Biodistribution Studies. Pilot 177Lu biodistribution studies in nude mice bearing LS174T human colon carcinoma xenografts were conducted to compare CC49 conjugates of DOTA and MeO-DOTA. Uptakes in the major organs of interest at 24 h postinjection are shown in Figure 3. For 177Lu-MeO-DOTACC49, substantially lower kidney uptake was observed, con177Lu-MeO-DOTA-CC49
Mohsin et al.
Figure 4. Biodistribution (n ) 5) of 149Pm-MeO-DOTA-CC49 in LS174T-bearing nude mice. Error bars represent standard deviations (SD).
Figure 5. Biodistribution (n ) 5) of 166Ho-MeO-DOTA-CC49 in LS174T-bearing nude mice. Error bars represent standard deviations (SD).
comitant with considerably higher LS174T tumor uptake and slower blood clearance. With 177Lu, the MeO-DOTA conjugate displayed superior conjugate and radiolabeling stability and more favorable biodistribution properties at 24 h postinjection. Therefore, MeO-DOTA-CC49 was selected for further in vivo evaluation using all three radiolanthanides. Comprehensive biodistributions of 149Pm- and 177Lu-MeODOTA-CC49 were obtained in the LS174T mouse model at 15 min, 1, 4, 24, 48, 96, and 168 h postinjection. Biodistributions for 166Ho-MeO-DOTA-CC49 were obtained at the same time points out to 96 h postinjection. The results obtained for 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49 are given in Figures 4, 5, and 6, respectively. MeO-DOTA-CC49 exhibited extremely high tumor uptake for each of the radiolanthanides. The highest tumor uptake of 166Ho-MeO-DOTA-CC49 was 69.5% ID/g at 96 h, which was significantly lower (p < 0.05) than the tumor uptake of 149Pm- and 177Lu-MeO-DOTA-CC49 (100.0% and 95.4%, respectively) at the same time point. Maximum tumor uptake of 177Lu-MeO-DOTA-CC49, 132.4% ID/g, was observed at 168 h postinjection. This value was significantly higher than the maximum uptake observed for 166Ho- (p ) 0.001) and 149Pm-MeO-DOTA-CC49 (p ) 0.001).
149Pm-, 166Ho-,
and
177Lu-Labeled
mAb CC49
Figure 6. Biodistribution (n ) 5) of 177Lu-MeO-DOTA-CC49 in LS174T-bearing nude mice. Error bars represent standard deviations (SD).
Normal organ uptakes were generally below uniform distribution, except for initial uptakes of 177Lu in the liver (63.3% ID/ g), spleen (45.3% ID/g), and kidney (30.0%). Nontarget organ uptakes of 177Lu decreased significantly (p < 0.05) with time to approximately 7% ID/g (kidney), 12% ID/g (spleen), and 20% ID/g (liver) at 96 to 168 h. Corresponding uptakes of 149Pm and 166Ho remained essentially constant at these values throughout the course of the studies. Blood levels of radiolanthanidelabeled MeO-DOTA-CC49 decreased significantly (p < 0.05) from approximately 21 to 27% ID/g at 3 h to 8 to 15% ID/g at 96 h. Bone uptakes of all three radiolanthanides decreased significantly (p < 0.05) from 4.89 to 7.66% ID/g at 15 min to 2.65 to 4.51% ID/g by 96 h. Small intestinal uptakes peaked at 1.33 to 2.52% ID/g at 6 h, and large intestinal uptakes reached maximum values of 1.33 to 2.48% ID/g by 6 h (data not shown). Tumor-to-Blood Time-Activity Ratios of 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49. The biodistributions of 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49 were used to generate tumor and blood time-activity curves for the three radioimmunoconjugates. Physical decay was included to calculate percent injected activity per gram of tissue (% IA/g). The ratio of areas under the curves (AUCs) was used to estimate the anticipated “therapeutic index” of each agent. For 149Pm-MeODOTA-CC49, the tumor AUC was 4405% IA/g-h, and the blood AUC was 1177% IA/g-h, affording a tumor-to-blood ratio of 3.74. Using 166Ho-MeO-DOTA-CC49, lower values were obtained, yielding a tumor AUC of 1329% IA/g-h, a blood AUC of 596% IA/g-h, and a tumor-to-blood ratio of 2.23. MeO-DOTA-CC49 labeled with 177Lu gave the highest results, with a tumor AUC of 9166% IA/g-h, a blood AUC of 1778% IA/g-h, and a tumor-to-blood ratio of 5.15.
DISCUSSION The present studies evaluated the anti-TAG-72 monoclonal antibody CC49 conjugated with two bifunctional chelating agents, DOTA-OSSu and MeO-DOTA, for labeling with lanthanide radionuclides. Over the past several years, numerous groups have developed a large number of C- and N-substituted analogues of DOTA for antibody conjugation. Using DOTAOSSu, we have had success targeting mouse xenografts with a variety of mAbs and radiometals (5-10). Conversely, MeODOTA may have better binding and in vivo distribution properties for radiolanthanides. A recent study compared antiTAG-72 mAb B72.3 conjugated to MeO-DOTA, an octadentate
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C-substituted DOTA derivative, and a heptadentate DOTA attached by conversion of one of the carboxylate groups to an amide linker (34). In non-tumor-bearing mice, 177Lu-labeled MeO-DOTA-B72.3 showed the lowest lung and spleen uptake of the three conjugates, suggesting reduced formation of colloidal radiometal hydroxides in vivo. Bone uptake of 177LuMeO-DOTA-B72.3 was lower than that of amide-linked DOTA and comparable to that of C-substituted DOTA, indicating high chelate stability. Blood clearance of 177Lu-MeO-DOTA-B72.3 was also faster than that of the C-substituted conjugate. Another advantage of MeO-DOTA is that it is the product of a convergent synthesis, which is considerably more straightforward than those of many C-substituted DOTA analogues. Several mAbs (cT84.66, cBR96, and NR-LU-10) have been conjugated with DOTA-OSSu to give immunoconjugates that labeled reproducibly to high specific activity and high stability with 111In, 90Y, and 64Cu (5-10). One preparation of DOTA-cT84.66 (5) has afforded near quantitative labeling and immunoreactivity after more than nine years of storage at 4 °C (8, 9). In contrast, the DOTA-CC49 conjugate was not stable for extended storage at 4 °C, and after approximately two months, 177Lu labeling gave consistently irreproducible results. This finding was unusual and suggested that the choice of mAb and bifunctional chelating agent may be important for developing stable and high specific activity radiolanthanide immunoconjugates for RIT applications. However, at present we have no logical explanation for the instability of DOTA-CC49 upon storage. Unlike the DOTA conjugate, MeO-DOTA-CC49 could be labeled with 149Pm, 166Ho, and 177Lu stably and with reproducibly high specific activity, even after more than nine months of storage at 4 °C. For initial comparisons of the in vivo properties of two 177Lu chelate conjugates, pilot biodistributions in the major organs were obtained for 177Lu-DOTA-CC49 and 177Lu-MeO-DOTACC49 in LS174T-bearing nude mice at 24 h postinjection. When labeled with 177Lu, MeO-DOTA-CC49 showed lower kidney uptake, slower blood clearance, and higher LS174T tumor uptake. For CC49, MeO-DOTA was a more robust bifunctional chelating agent for stable conjugation and reproducible, efficient labeling than DOTA. Therefore, MeO-DOTA-CC49 was chosen for further evaluation in vitro and in vivo using 149Pm, 166Ho, and 177Lu. To assess the in vitro stability of radiolanthanide-labeled CC49, studies were conducted in mouse serum and a hydroxyapatite model. In serum, the small degree of radiometal dissociation from the conjugate resulted only in lower molecular weight constituents in serum out to 96 to 168 h at 37 °C, consistent with other published reports evaluating 111In-, 90Y-, and 64Cu-labeled mAbs (5, 6, 8-10). Relative to initial radiochemical purity, the serum stability of 177Lu-DOTA-CC49 was statistically equivalent to 177Lu-MeO-DOTA-CC49 over a 96-h period. Only at 168 h was the MeO-DOTA conjugate more stable than the DOTA conjugate when labeled with 177Lu. Because of the lanthanide contraction, lutetium has the smallest radius and highest coordination energy of the three radiometals and is likely to be bound more tightly by the MeO-DOTA chelator. MeO-DOTA-CC49 was most stable in serum when labeled with 177Lu, followed by 166Ho and 149Pm. However, radiolanthanides do not bind with high affinity to any major serum proteins. At longer time points in the serum stability studies, equilibria likely exist between the radiolanthanide chelate conjugates and the free radiometals. Over an extended period, the serum stability curves began to reach asymptotes for all three radioimmunoconjugates. This asymptotic behavior was characteristic of equilibrium conditions, under which thermodynamic stability of the radiolanthanide-mAb conjugates would be expected to prevail.
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However, the in vivo stability of radiolanthanide-labeled CC49 is likely to depend on kinetic, rather than thermodynamic, properties. Therefore, a hydroxyapatite matrix (32) was used as an in vitro model to predict the relative in vivo stabilities of 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49. Hydroxyapatite is a major constituent found in the extracellular matrix of bone. As calcium mimics, free lanthanides deposit and become fixed in bone, presumably by a hydroxyapatite-mediated mechanism. In contrast to the serum stability studies, hydroxyapatite binding showed that 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49 were g92% stable for up to 168 h at 37 °C. The difference in stability of 149Pm-, 166Ho-, and 177Lu-labeled CC49 in hydroxyapatite versus serum may reflect differences in kinetic versus thermodynamic stability, as measured respectively by the two assays. Dissociation of radiolanthanides from mAb conjugates in vivo will likely result in substantial accumulation in the skeleton. In the present studies, the high stabilities of 149Pm-, 166Ho-, and 177Lu-MeO-DOTA-CC49 to hydroxyapatite challenge paralleled the low bone uptakes of the three radiometals in LS174Tbearing nude mice. Bone uptakes were not statistically significant between the radiometals (p > 0.05), and by 96 h values for all three radiolanthanide immunoconjugates were 149Pm > 166Ho. In subsequent RIT studies (36), it was determined that the most significant endpoint was time to progression to an LS174T tumor burden of 1 g. Using this endpoint, it was found that the therapeutic efficacy of 177Lu-MeO-DOTA-CC49 was statistically superior to that of the 149Pm conjugate, which in turn was statistically greater than that of the 166Ho conjugate. These results suggest that, given the relatively long tumor targeting and clearance kinetics of intact radiolabeled antibodies, the physical half-life of the radionuclide should be well matched to the biological half-life of the targeting agent.
CONCLUSION MeO-DOTA-CC49 labeled with the lanthanide radionuclides and 177Lu showed extremely high tumor uptake and generally favorable biodistribution properties in nude mice bearing LS174T human colon carcinoma xenografts. The high tumor-specific targeting of each of these agents warrants further investigation for RIT of adenocarcinomas. However, the different physical half-lives and β- particle energies of the radiolanthanides, as well as the different clearance properties of the radioimmunoconjugates, may have important effects on radiation dosimetry, toxicity, and therapeutic efficacy. The results of RIT studies of 149Pm-, 166Ho-, and 177Lu-MeODOTA-CC49 in the LS174T mouse model have been presented in preliminary form (36) and will be reported in more detail soon. 149Pm, 166Ho,
ACKNOWLEDGMENT This research, under Award Number DAMD 17-02-1-0103, was supported by the Department of Defense Prostate Cancer Research Program, which is managed by the U.S. Army Medical Research and Materiel Command. This work was also funded by Grant URB-01-015 from the University of Missouri Research Board and a grant from the University of Missouri College of Veterinary Medicine Committee on Research. The authors would like to thank Dr. Jeffrey Schlom for providing the CC49 hybridoma cell line, as well as Dr. Christopher Johnson for assistance with statistical software SPSS 12.0.1. We also acknowledge the support of the U.S. Department of Veterans Affairs, for providing resources and the use of facilities at the Harry S. Truman Memorial Veterans’ Hospital in Columbia, MO. Supporting Information Available: Tables of mean percent injected dose per gram of tissue (% ID/g) with standard deviations (SD) of 149Pm-, 166Ho-, and 177Lu-MeO-DOTACC49 for all tissues and time points evaluated. This material is available free of charge via the Internet at http://pubs.acs.org.
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