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An Improved Method for Labeling Monoclonal Antibodies with Samarium-153: Use of the Bifunctional Chelate 2- (p-Isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic Acid M. E. Izard,? G.R. Boniface,*J K. L. Hardiman,? M. W. Brechbiel,* 0. A. Gansow,* and K. Z. Walkers8 Biomedicine and Health Program, ANSTO, Lucas Heights, New South Wales, Australia, Chemistry Section, National Cancer Institute, NIH, Bethesda, Maryland, and Centenary Institute for Cancer Medicine and Cell Biology, Sydney, New South Wales, Australia. Received April 10, 1992 Samarium-153 ( 9 3 m ) radioimmunoconjugates of the monoclonal antibody K-1-21 were produced using the bifunctional chelate 2-@-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic acid (MxDTPA). The specific activity (up to 150 MBq mg-l) and percent retained immunoreactivity (>75%) were similar to that of 153Sm-K-1-21conjugates formed with cyclic DTPA anhydride (cDTPAa). In vivo biodistribution studies showed specific localization of 153Sm-Mx-DTPA-K-1-21 to target antigen implants and higher blood pool and lower uptake in liver, spleen, kidney, and bone when compared to '53Sm-cDTPAa-K-1-21. The improved in vivo distribution of 153Sm-Mx-DTPA-K-1-21 should result in lower radiotoxicity to nontarget tissues when used for radioimmunotherapy purposes.
INTRODUCTION Samarium-153 (15%m)is a 0-la-emitting radionuclide of intermediate half-life and 0 energy which may be appropriate for the production of radioimmunoconjugates with various antitumor monoclonal antibodies (mAb) for radioimmunotherapy. We have previously reported the successfullabeling of the monoclonal antibody K-1-21with 15%m using the bifunctional chelate cyclic DTPA dianhydride (cDTPAa) at specific activities up to 150 MBq mg' (1). The mAb K-1-21 binds to free monomers and dimers of human K light chains (LC) but not heavy-chainassociated light chains. It also recognizes a tumorassociated antigen on some K myeloma and K lymphoma cells (2,3).Biodistribution studies of 15%3m conjugates of the mAb K-1-21 indicated specific localization to subcutaneous implants of the K antigen covalently linked to Sepharose 6MB CNBr-activated beads (Pharmacia) in a rat tumor model system to a similar degree to W- or lllIn-labeled K-1-21. However, significant radioactivity was also noted in liver, kidneys, and osteous bone. In addition, serum levels of radioactivity were significantly lower than 1311-or lllIn-labeled K-1-21 indicating some in vivo dissociation of the radiolabel (I). A similar biodistribution pattern was also observed when la3Smconjugates of the anti-bladder cancer mAb BLCA-38 were injected systemically into nude mice bearing tumor xenografts of the human bladder cancer cell line BL17 (4). Although the 15%m-BLCA-38conjugates showed significant tumoricidal activity, a dose-limiting radiotoxicity to liver and bone marrow was calculated from dosimetric estimations (5).
The synthesis of a number of diethylenetriaminepentaacetic acid based bifunctional chelating agents containing an isothiocyanato benzyl group as protein linker have been described (6). The synthesis of the chelating agent used here was described by Brechbiel (7)and Brechbiel
* Corresponding author: Graeme R. Boniface, BIOMIRA INC, Research Centre One, Edmonton Research & Development Park, 9411-20Ave, Edmonton, Alberta, Canada T6N 1E5. + ANSTO. 1 NIH. f
Centenary Institute for Cancer Medicine and Cell Biology. 1043-7002/92/ 2903-O346$O3.00/0
and Gansow (8)as Mx-DTPA [2-@-isothiocyanatobeenzyl)6methyldiethylenetriainepentaaceticacid1 and its structure has been confirmed by Cummins et al. (9). This bifunctional chelator has recently been shown to improve the in vivo stability of both lllIn and mAb conjugates ( 1 0 , I I ) . In this study we have investigated the suitability of Mx-DTPA as a bifunctional chelator for preparing 15%m-mAb conjugates. In addition the biodistribution of Wm-Mx-DTPA-K-1-21 was compared with that of 153Sm-cDTPAa-K-1-21in the K antigen implant rat tumor model system (12). The number of chelator molecules per antibody molecule was determined with 14C intrinsically labeled Mx-DTPA (7). EXPERIMENTAL PROCEDURES Monoclonal Antibody Production andPurification. The IgGl mAb K-1-21 was purified from ascites by ammonium sulfate fractionation and affinity chromatography on ROW K LC conjugated to Sepharose CL-4Bbeads (Pharmacia, Uppsala, Sweden). Purified antibody was suspended in phosphate-buffered saline, pH 7.2 (PBS),at 14.8 mg mL-l and stored at -20 "C. Preparation of la3Sm. Samarium oxide (152Sm203) enriched to 98.7 % purity was activated (n,r) in a neutron flux of 5 X 1013n cm-2 s-l in the ANSTO HIFAR reactor to a specific activity of 31 GBq mgl. The activated oxide was dissolved in 6 N HC1 and evaporated to dryness. The 153SmC13was dissolved in ultrapure water to a radioactive concentration of 18.5 GBq mL-l at pH 5.3. 15%m was used as the citrate salt at pH 7 following dilution with 0.2 M sodium citrate, pH 7, to a specific activity of 10 GBq mL-1. Cyclic DTPA Anhydride Conjugation to mAb. Cyclic DTPA anhydride (cDTPAa) (Sigma Chemical Co., St. Louis, MO) was conjugated to K-1-21 (14.8 mg mL-1) at a reactive molar ratio of 201 cDTPAa:K-1-21 as previously described (I). The cDTPAa-K-1-21 was separated from unconjugated DTPA by centrifugal sizeexclusion chromatography (13)and buffer exchanged into 0.2 M, pH 7 citrate buffer on Bio-Gel P-6DG (Bio-Rad, Richmond, CAI. Mx-DTPA Conjugation to mAb. Mx-DTPA or 14CMx-DTPA was freshly dissolved in ultrapure water and reacted with K-1-21 at Mx-DTPA:K-1-21 molar ratios @ 1992 American Chemical Society
Tschnlcal Notes
between 2.51 and 2 5 1 in 0.14 M phosphate buffer, pH 9 (6, 13,14). Concentration of the antibody in the conjugation reaction was 7 mg mL-l. The reaction was maintained at pH 9 and incubated at 37 OC for 2.5 h, prior to purification and buffer exchange into 0.2 M, pH 7 citrate buffer. The number of chelator molecules per antibody molecule was determined by liquid scintillation counting of the purified 1%-Mx-DTPA-K-1-21 conjugate and 14C-Mx-DTPAstandards suspended in scintillation fluor (Instagel, Packard). Labeling of cDTPAa-K-1-21 and Mx-DTPA-K- 121 with l%m. Fifty megabecquerel of 153Sm(10 GBq mL-l in 0.2 M, pH 7 citrate buffer) was added to the purified cDTPAa- or Mx-DTPA-conjugated K-1-21 (200 pg) and incubated a t room temperature for 30 min. The labeled antibody was then purified by either centrifugal size-exclusion chromatography or by elution from a 20 mm X 150 mm P-6DG (Bio-Rad) size-exclusion column. The purified conjugateswere adjusted to a volume of about 300 pL with citrate buffer (0.01 M, pH 7) in a Centricon30 microconcentrator (Amicon). Citrate buffer (500 pL, 0.01 M, pH 7) was added to the microconcentrator and the volume reduced to about 300 pL by centrifuging a t l600g for 8 min. Thiswashing procedure was repeated four times. The conjugates were finally washed in sterile physiological saline before injection into animals. Total activity retained on the antibody was determined after each wash with an isotope dose calibrator. Nonspecific Binding of l%m to K-1-21. Stock K1-21(200pg) in PBS was buffer exchanged viaa centrifugal P-6DG column into0.2 M, pH 7 citrate buffer. The column eluent was incubatedwith samarium-153citrate (50 MBq, 10 GBq mL-l) for 30 min and then purified through either a 20 mm X 150 mm or a centrifugal P-6DG gel column. Activity in the second column eluent was measured with an isotope dose calibrator. Immunoreactivity of lS3Sm-K-l-2l. The immunoreactivity of the 153Sm-K-1-21conjugates was assessed by a solid phase binding assay to immobilizedK LC Sepharose beads as previously described (1,12). HPLC Analysis of laSm-K-l-21. Size-exclusion HPLC (BiosilTSK 250 (Bio-Rad),0.2 M Tris, pH 7.2) was performed on representative samples of the labeled conjugates as previously described (1). Biodistribution Studies. Male Fisher 334 rats aged between 10 and 15 weeks were used for biodistribution studies. K (test)and X (control) LC-conjugated Sepharose beads (ca. 0.5 mL) were implanted into opposite flanks of the animals as previously described (12). Twenty four hours after the placement of implants, groups of five animals were injected ip with 10-20 pg (0.3-1 MBq) of lS3Sm-cDTPAa-K-1-21 or '53Sm-Mx-DTPA-K-1-21. Six days after injection each animal was exsanguinated, dissected, and selected organs counted in a y counter (Riagamma, LKB/Wallac, Sweden). After 6 days each gel implant was covered by a thin vascularized membrane which facilitated its removal as an intact capsule. These gel capsules were cleared of fat and connective tissue before weighing and counting. Tissue distribution profiles of percent injected dose per gram (%IDgl),percent injected dose per organ (5% ID), tissue:blood ( T B ) ratio, implankblood ratio, and K:A specificity index were calculated using a computer biodistribution program. Data were compared by Student's t-test.
RESULTS ConjugationofMx-DTPAtoK-1-11. Theconjugation of the bifunctional chelate Mx-DTPA to the K-1-21 mAb
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Table I. Conjugation of WC-Mx-DTPA to K-1-21 Mx-DTPAK-1-21 s ecific activity % retained mole ratio of incorporationn 0!~Wm-K-1-21 immune Mx-DTPAK-1-21 (mokmol) (MBq mgl)* reactivity 251 6.3 130 a7 101 2.4 56 a5 51 1.9 44 88 2.51 0.2 24 a6 Reacted for 2.5 h at 37 O C . Using samarium-153 citrate at 31 GBq m g l . Table 11. Effect of W m Concentration on Specific Activity of Wm-Mx-DTPA-K-1-21 mole ratio of 153Sm: specific activity of "Sm-Mx-DTPA-K-1-21 MX-DTPA-K-1-21 before wash after wash ~
121 0.6:l
133 37
~~
117 30
was studied using 14C labeled Mx-DTPA and liquid scintillation counting. A near linear relationship was observed between the amount of 14C incorporated onto the mAb and the Mx-DTPAK-1-21molar ratio employed (Table I). At a starting Mx-DTPAK-1-21 molar ratio of 2.5:1, an average of 0.2 mol of Mx-DTPA were added to the mAb within 2.5 h. At a Mx-DTPAK-1-21molar ratio of 25:1, an average of 6.3 mol of Mx-DTPA was added. These were the reaction conditions selected for subsequent conjugation preparations for biodistribution studies using unlabeled Mx-DTPA bifunctional chelate. Conjugation of cDTPAa to mAb. The conjugation of cDTPAa to mAb at various ratios of cDTPAa:K-1-21 and HPLC analyses of the labeled conjugates have been previously described (I). The number of cDTPAa molecules attached to the mAb was not quantified, but at the cDTPAa:K-1-21 ratio of 20:1, chosen for the biodistribution studies, there was minimal cross-linking of the conjugate and immunoreactivity remained at about 90 5% after preparation. Preparation of lBSm-Mx-DTPA-K-1-21. Successful labeling of K-1-21 occurred with the use of Mx-DTPA as bifunctional chelate. The specific activity of the labeled conjugate depended on both the mole ratio of Mx-DTPA: K-1-21 in the conjugation reaction and the total activity of the 153Sm in the chelation reaction. Using excess samarium-153 citrate with specific activity of 31 GBq mg-l and Mx-DTPA:K-1-21 ratios of between 2.5:l and 25:l produced '53Sm-Mx-DTPA-K-1-21 conjugates with specific activities between 24 and 130 MBq m g l (Table I). Size exclusion purified Mx-DTPA-K-1-21 conjugate (Mx-DTPA:K-1-21, 25:l) labeled a t 153Sm:K-1-21mole ratios of 12:l and 0.6:l produced labeled conjugates with specific activities of 133 and 37 MBq mg-l before citrate wash and 117 and 30 MBq m g l after three washes (Table 11). Retained immunoreactivity, determined by solid phase binding assay, remained above 80% for these preparations. When the mole ratio of Mx-DTPA:K-1-21 in the conjugation reaction was raised to 50:l the specific activity of the labeled conjugate produced was increased to 230 MBq mg-'; however, immunoreactivity decreased to 64%. Nonspecific Binding of ls3Smto K-1-21. No '53Sm was detected in the column eluent containing the antibody when unconjugated K-1-21 incubated with 153Sm was purified through the 20 mm X 150mm size-exclusionchromatography column. Less than 0.5% of total activity added to the unconjugated K-1-21was recovered following purification by the centrifugal column technique. Citrate Wash of Labeled Conjugates. Following purification of the labeled antibody through size-exclusion
348 Bloconlugete Chem., Vol. 3, No. 4, 1992
Izard et al. 60
~
113 cDTPAa (unwashed)
A 50
A
cDTPAa ( w a W
-
Mx.DTPA (unwarhed) Mx.DTPA (waskd)
40
-
30
-
KAPPA LAMBDA
10
5
LV
KD
LU
MM
SP
SK
ST
bL
BO
15
Retention Time (min) 14
12 10
B
6 6 4
2 0
KAPPA
LAMBDA
LV
LU
KD
MM
SK
SP
ST
BO
Figure 2. Biodistribution of T3m-K-1-21 in Fisher rata 6 days postinjection (mean SEM,n = 5); (A) percent injected dose per gram, (B) tissue:blood ratio. Abbreviations: KAPPA = K antigen implant, LAMBA = h antigen implant, LV = liver, LU = lung, KD = kidney, MM = muscle, SK = skin, SP = spleen, ST = stomach, BL = blood, BO = bone.
*
The resultant K implant uptakes were 1.9 f 0.2 and 3.6 f 0.1 % ID g-1 for unwashed and washed '53Sm-cDTPAaK-1-21 and 4.67 f 1.0 and 4.45 f 0.9% ID g1for Ia3Sm-
. I
abSwbmC8 1 .-__--_____.. I
5
\
-
-
.._----. t.
I
6
10
15
Retention Time (min)
Figure 1. Size-exclusion HPLC chromatograms of gel column purified "Sm-Mx-DTPA-K-1-21 (A) before and (B) after citrate wash. Retention time of 158Sm-Mx-DTPA-K-1-21 at 9.68 min, non-antibody-bound 153Sm at 13.8 min (Biosil TSK-250, Tris buffer 0.2 M, pH 7.2, 1 mL/min).
columns,HPLC analysis revealed the antibody to be clearly labeled with minimal cross-linking (Figure 1). It also showed the presence of some l53Sm activity not associated with the antibody. This activity was completely removed by the citrate washing. Biodistribution Studies. Biodistribution studies confirmed that both Wm-cDTPAa-K-1-21 and 153Sm-MxDTPA-K-1-21 exhibited specific localization to the K antigen implants at 6 days postinjection.
Mx-DTPA (Figure 2). The K:X (control implant) specificity index was 2.0 and 3.76 for unwashed and washed lass,cDTPAa-K-1-21 and 3.85 and 3.42 for unwashed and washed 153Sm-Mx-DTPA-K- 1-21. Blood retention for the unwashed 153Sm-cDTPAa-K1-21was 4.2 f 0.6% ID and 5.09 f 0.7% ID for the washed preparation. Both 1MSm-Mx-DTPA-K-1-21 preparations showed increased blood retention with 9.38 f 1.8%ID for the unwashed and 10.46 f 2.0% ID for the washed. This increased blood retention resulted in reduced tissue:blood ratios in all organs in comparison to the 153Sm-cDTPAaK-1-21 preparations (Figure 2). More importantly however, the implant:liver, implant:kidney, and implantbone ratios for both unwashed (3.5, 7.1, 7.9) and washed (6.3, 9.9, 18.5) 153Sm-Mx-DTPA-K-1-21 were significantly higher than those seen with the 153Sm-cDTPAa-K-1-21 conjugate both unwashed (1.5,0.9, 2.3) and washed (2.0, 2.4, 1.7). DISCUSSION
The potential of radioimmunotherapy of tumors by the use of systemically administered &emitting radionuclidemAb conjugates has been limited to date due to a number
Technical Notes
of factors associated with the nonspecific accumulation of radioimmunoconjugate in nontarget tissues, particularly the liver and bone (1, 13, 15). This uptake has been particularly prevalent when radiolanthanides, such as yttrium or samarium, have been conjugated to mAbs via cyclic DTPA anhydride. The natural predilection of intact mAbs to be metabolized in the liver (161,combined with significant transchelation of radionuclide to subcellular components within the hepatocyte, has resulted in unacceptably high liver dose predictions a t therapeutic dose levels. In addition, the natural bone-seeking properties of yttrium, and to a lesser extent samarium, ions have resulted in significant transchelation of these radionuclides from the mAb to osteous bone matrix, with resultant high radiation doses to neighboring marrow. This is of particular concern with wY due to the higher penetration of the more energetic 8 emissions of this radionuclide. In order to reduce the nontarget uptake of radiolabeled mAbs for radioimmunotherapeutic purposes, several alternative regimens have been proposed including the use of metabolizable linkers, antibody fragments, or heterobifunction antibodies, with radiochelate chase (13,15). Other workers have proposed the use of macrocycle bifunctional chelates to enhance binding of metal ions to mAbs (17,18). While it is still conjecture whether enhancing the stability of the radionuclide-mAb attachment will alone diminish the hepatic transchelation of some metal ions, there is clear evidence that bone transchelation can be minimized. The use of bifunctional chelates which elicit improved chemical stability of the radionuclide to the mAb by retaining the denticity of the DTPA moiety have recently been shown to reduce the “off rate” of radiolabel from the mAb in vivo. Using a series of backbonesubstituted DTPA analogues (Mx-DTPA, 1M3B-DTPA, 1B3M-DTPA) Kozak and colleagues have shown significant increases in the in vivo stability of 111In and MY labelled anti-Tac mAb in comparison to cDTPAa conjugates (10).A reduction in bone uptake, together with a reduced plasma clearance of labeled anti-Tac, was seen using these derivatives in comparison to cDTPAa. However, liver uptake remained unaffected. Our studies extend these findings by demonstrating that the biodistribution of 153Sm labeled mAb in animals bearing model tumor implants is improved by using the DTPA derivative 2-@-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic acid rather than the cyclic DTPA anhydride. A reduction in bone uptake and increased retention in the blood was observed with 153Sm-Mx-DTPA-K-1-21. Despite these improvements the liver uptake of 153Smwas not significantly reduced for Mx-DTPA conjugates of K-1-21 purified by conventional gel chromatography methods only. HPLC analyses of these size-exclusion-purified conjugates revealed, along with the labeled antibody, some activity unassociated with the antibody. The activity was not nonspecifically bound 153Smbut may have been due to postpurification release of 153Smweakly chelated to conjugated Mx-DTPA. Dissociation in vivo from the conjugate of any weakly chelated 153Smnot removed by the purification regime would contribute to liver uptake seen in the biodistributions of those conjugates without the citrate wash. The purification regime with citrate buffer of 153Sm-Mx-DTPAK-1-21 produced a conjugate whose liver uptake diminished to the level of the blood pool. The Mx-DTPA bifunctional chelate appears to be an attractive alternative to cDTPAa for producing 153Sm-
Bioconjwate Chem., Vol. 3, No. 4, 1992 349
mAb radioimmunoconjugates yielding reduced osteous deposition and improved blood retention. Further studies are continuing with 153Sm-Mx-DTPA conjugates of the anti-bladder mAbs BLCA-38and BLCA8 for the intravesical and systemic radioimmunotherapy of bladder cancer in cancer xenograft animal models (2, 3). ACKNOWLEDGMENT
We thank Mr. P. Sorby and D. Henderson of ANSTO for the production of 153Sm. The expert technical assistance of Ms. Sharon Parkes of ANSTO with HPLC and immunoreactivity estimations is also gratefully acknowledged. LITERATURE CITED (1) Boniface, G. R., Izard, M. E., Walker, K. Z., McKay, D. R., Sorby, P. J., Turner, J. H., and Morris, J. G. (1989) Labeling of monoclonal antibodies with samarium-153 for combined radioimmunoscintigraphy and radioimmunotherapy. J . Nucl. Med. 30,683-691. (2) Boux, H. A., Raison, R. L., Walker, K. Z., Hayden, G. E., and Basten, A. (1983) A tumour-associated antigen specific for human kappa myeloma cells. J . Exp. Med. 158, 1769-1794. (3) Goodnow, C. C., and Raison, R. L. (1985) Structural analysis of the myeloma-associated membrane antigen KMA. J . Immunol. 135, 1276-1280. (4) Walker, K. Z., Boniface, G. R., Lightfoot, D. V., Ormsby, S., Izard, M. E., Parkes, S. L., Weedon, A., and Russell, P. J. (1990) Samarium-153-labeled monoclonal antibody BLCA38. I. Biodistribution studies in a nude mouse xenograft model. Antibodies, Zmmunoconjugates Radiopharm. (submitted). (5) Lightfoot, D. V., Walker, K. Z., Boniface, G. R., Hetherington, E. L., Izard, M. E., and Russell, P. J. (1991) Dosimetric and therapeutic studies in nude mice xenograft models with samarium-153labelled monoclonal antibody. Antibodies,Zmmunoconjugates Radiopharm. 4 (3), 319-330. (6) Brechbiel, M. W., Gansow, 0. A., Atcher, R. W., Schlom, J., Esteban, J.,Simpson, D. E., and Colcher, D. (1986) Synthesis of 1-(p-Isothiocyanatobenzyl)derivativesof DTPA and EDTA. Antibody labeling and tumor-imaging studies. Znorg. Chem. 25,2772-2781. (7) Brechbiel, M. W. (1988) New bifunctional ligands for radioimmunoimaging and radioimmunotherapy. Ph.D Thesis, The American University, Washington, D.C. (8) Brechbiel, M. W., and Gansow, 0. A. (1991) Backbonesubstituted DTPA ligandsfor Y-90 radioimmunotherapy. Bioconjugate Chem. 2, 187-194. (9) Cummins, C. H., Rutter, E. W., and Fordyce, W. A. (1991) A convenient synthesis of bifunctional chelating agents based on diethylenetriaminepentaacetic acid and their co-ordination chemistry with yttrium (111). Bioconjugate Chem. 2, 180186. (10) Kozak,R. W.,Raubitschek,A., Mirzadeh,S.,Brechbiel,M. W., Junghaus, R., Gansow, 0. A., and Waldmann,T. A. (1989) Nature of the bifunctional chelating agent used for radioimmunotherapy with yttrium-90 monoclonalantibodies: Critical factors in determining in vivo survival and organ toxicity. Cancer Res. 49, 2639-2644. (11) Roselli, M., Schlom, J., Gansow, 0. A., Raubitschek, A., Mirzadeh, S., Brechbiel, M. W., and Colcher, D. (1989) Comparative biodistributions of yttrium- and indium-labeled monoclonal antibody B72.3 in athymic mice bearing human colon carcinoma xenografts. J . Nucl. Med. 30, 672-682. (12) Walker, K. Z., Seymour-Munn, K., Keech, F. K., Axiak, S. M., Bautovich, G. J., Morris, J. G., and Basten, A. (1986) A rat model system for radioimmunodetection of kappa myeloma antigen on malignant B cells. Eur. J . Nucl. Med. 12,461-467. (13) Meares, C. F., McCall, M. J., Reardan, D. T., Goodwin, D. A., Diamanti, C. I., and McTigue, M. (1984) Conjugation of antibodies with bifunctional chelating agents: Isothiocyanate and bromoacetamide reagents, methods of analysis and subsequent addition of metal ions. Anal. Biochem. 142,6878.
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(14) Meares, C. F.,and Goodwin, D. A. (1984)Linking radiometals to proteins with bifunctional chelating agents. J.B o t . Chem. 3 (2), 215-228. (15) Deshpande, S. V.,DeNardo, S. J., Meares, C. F., McCall, M. J.. Adams. G. P.. and DeNardo. G. L. (1989) Effect of different linkages between chelates and monoclonal antibodies on levels of radioactivity in the liver. J. Nucl. Med. Biol. 16, 587-597. (16) Sands, H.,and Jones, P. L. (1987)Methods for the study of the metabolism of radiolabelled monoclonal antibodies by liver and tumor. J. Nucl. Med. 28, 390-398.
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(17) Craig, A. S.,Helps, I. M., Jankowski, K. J., Parker, D., Beeley, N. R. A., Boyce, B. A., Eaton, M. A. W., Millican, A. T., Millar, K., Phipps, A., Rhind, S. K., Harrison, A,, and Walker, C. (1989)Towards tumour imaging with indium-111 labelled macrocycle-antibody conjugates. J. Chem. SOC.Chem. Commun. 12, 794-798. (18) Moi, M. K.,Meares, C. F., and DeNardo, S. J. (1988)The peptide way to macrocyclic bifunctional chelating agents: Synthesis of 2-(p-Nitrobenzyl)-l,4,7,lO-tetraazacycloddecaneN,N,N”,N”’-tetracetic acid and study of its yttrium (111) complex. J. Am. Chem. SOC.110,6266-6267.
