CaNa2EDTA for Improvement of Radioimmunodetection and Radioimmunotherapy with 1111n 90YDTPA-Anti-CEA and MAbs in Nude Mice Bearing Human

Colorectal Cancer
Naoyuki Watanabe, Noboru Oriuchi, Keigo Endo, Tomio Inoue, Masahide Kuroki, Yuji Matsuoka, Shuji Tanada, Hajime Murata, E. Edmund Kim, and Yasuhito Sasaki
Division ofAdvanced Technologyfor Medical Imaging, National Institute ofRadiological Sciences, Chiba; Department of Nuclear

Medicine, Gun,na University School ofMedicine, Gunma; Department ofBiochemistry, Fukuoka University School of Medicine, Fukuoka, Japan; and Department ofNuclear Medicine, University ofTexas M.D. Anderson Cancer Center@ Houston, Texas

111thand 90Ywith monoclonal antibodies (MAbs) may play a significant role in RID and RIT of various malignant or @Y-MAb, onthebiodistnbution ofradioactive isotopes innude diseases.These radioactive metals are necessary as an appro mice bearing human colon adenocarcinoma LS 180 tumor priatechelatorolabelMAb. Diethylenetriamine t pentaacetic expressing CEA, or human pulmonary carcinoma PC 9 tumor dianhydride (DTPA-anhydride) has been used as a chelator expressing no CEA, were then examined. As a control, 0.9% NaCI was used in both the in vitro and in vivo studies. Results: for covalent attachment to an MAb and for long-term CaNa2EDTA didnotcauseanydecreaseinlevelsof radioactivity radionuclide labeling (24). owever, release of 1In H from of radiolabeledMAbs. Pre- and post-treatmentwith CaNa2EDTA in blood may result in transport by reducedradioctivityin both specificand nonspecifictumors at 72 transferrin to the liver and other organs of the reticuloendo h after 111ln-MAb injection resulting in an increaseof the specific thelial system. The liver uptake poses a significant problem tumor-to-nonspecifictumor radioactivity ratio. The levels of he in patic and renal radioactivitywere also subsequentlydecreased that drastically limits the usefulness of In clinical trials, by CaNa2EDTA.On the other hand, CaNa2EDTApre- and because many carcinomas metastasize to the liver (1,5). post-treatmentreducedlevelsof bony,hepatic,and renalradioac Bone marrow activity may also interfere with the detection tivity at 24, 72, and 72 h, respectively,after @Y-MAb injection, althoughit had no effecton tumor radioactivity.Conclusion: Pro of bone metastases (6). Renal radioactivity of @In-MAb and post-treatment with CaNa2EDTAwould be of great use in may cause artifacts in the upper abdomen and make routine humans who undergo RID or AlT with 111ln-MAb and @Y-MAb clinical use more problematic. On the other hand, 90Y,leaked accompaniedby disassociationof the labeledradionuclides. from @Y-DTPA-MAb blood, can accumulate in bone, in KeyWords:111ln; calcium soY; disodium ethylenediaminetetraace because 90Yis itselfa bone-seekinggent(1,7). The bone a tic acid; radioimmunodetection;radioimmunotherapy uptake may cause myelosuppression. To improve RID and

111ln nd 90Y, issociatedfrom 111ln-labeled-monoclonal a d antibody (MAb) and @Y-labeIed may cause deterioration the MAb, of image quality in radioimmunodetection (RID) and undesirable irradiation of nontargeted tissue in radloimmunotherapy(All), respecthiely. aIm of this studywas to investigateany improve The ment in RID and AlT with 111ln-MAbnd @Y-MAbpre- and a by postadministrationf calciumdisodiumethylenetriaminetetraacetic o acid (CaNa2EDTA).Methods: Munne MAb F33-104 against carcinoembryonicantigen (CEA)was labeledwith 111In r @Y o by the diethylenetnamine pentaacetic (DTPA)-anhydridemethod. The influence of CaNa2EDTAon loss of radioactivity from 111ln-MAb @Y-MAb serum was investigated in vitro. The or in effects of CaNa2EDTA,administeredbefore and after 111In-MAb

dioimmunodetection (RID) involvesfindingbiologic

markers in malignant tumor cells and may serve as a confirmatory test for growing neoplasms. It can also serve to qualify a patient for radioimmunotherapy (RIT) using the same antibody but a different isotope (1). In a y emitter is at 171 and 245 keV, with a half-life of 67.9 h. 90Y is a @3 emitter at a maximum energy of 2.3 MeV, with a half-life of 64.0 h. 90Yis known to have certain radiolabeling character

isticsin commonwith WIn. Therefore,a combination of

J NucIMed2000;41:337-344

RIT with ll1@ and 90Y-DTPA-MAbs,several strategies have

been investigated, including the use of avidin-biotin labeling

techniqueseitherbiotinylated ( antibodies ndIlIn-labeled a

Received Jan.19,1999;revision accepted Jun.21, 1999.
For correspondence or reprints contact Naoyuki Watanabe, MD, PhD, Division of Advanced Technology for Medical Imaging, National Institute of
Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan.

avidin or streptoavidin, or streptavidin-conjugated MAb followed by injection of radiolabeled biotin) (811),to develop metal chelates that remain intact during the catabo lism of conjugated MAt,, and thus ensure the excretion of

thelabeledspecies (1216), to combine or with autologous

Ei@cr

OF EDTA ON RID

@r@r RIT atanabe W

et al.

337

gated chelate molecules by Sephadex G25 gel filtration (Pharma cia, Uppsala, Sweden) using 0.1 mol/L NaHCO3 as an eluant and was dialyzed in 50 mmoIIL phosphate-buffered saline (PBS), pH chelates withdivalent ndtrivalent a metals. ccessible etal 7.5, at 4C 24 h. For In A m for labeling, 500 pigDTPA-conjugated ions (both exogenous and endogenous) with a higher affinity MAb in 0.1 mol/L sodium acetate buffer, pH 5.8, was mixed with for CaNa2EDTA than calcium will be chelated, mobilized, In-chloride74 MBq/mL; Nihon Medi-Physics Co. Ltd., Nishi ( and excreted (18). CaNa2EDTA has been reported to facili nomiya, Japan) and allowed to react at room temperature for 60 mm. After the addition ofdisodium salt EDTA(Na2EDTA; DOJIN) tate elimination of @In from mice bearing human tumor the cells prelabeled with In-oxine (19). It also has been shown to a final concentrationof 5 mmol/L to chelateunboundSIn In-MAb purified by Sephadex G25 gel filtration using 0.1 was that the administration of CaNa2EDTA substantially in mol/L sodium acetate buffer, pH 5.8. For @Y labeling, 500 jig creased the tumor-to-muscle radioactivity ratio in nude mice DTPA-conjugated MAb in 0.1 mi/L sodium acetate buffer, pH 5.8, injected with In-labeled (20). CaNa2EDTA MAb hasbeen was mixed with @Y-acetate MBq/mL), which was obtained by (925 shown in human subjects and mice to accelerate 90Y incubation of an equal volume of 1 mol/L sodium acetate buffer, pH excretion by chelating the released 9Y without compromis 5.8, and @Y-chloride (Nordion; Kanata, Ontario, Canada) for 30 ing the tumor uptake of 9Y-labeledMAb and to allow an mm at 37C allowed to react at room temperature for 60 mm. and

bone marrow transplantation (17). A few chelates for labeling may have immunogenic properties (5, 16). Calcium disodium ethylenetriaminetetraacetic acid (CaNa2EDTA), which has been clinically used for treatment of heavy metal poisoning, may eliminate several metallic radionuclides from the body effectively. The pharmacologic effects of CaNa2EDTA result from the formation in the body of

labelingof thecrudemixture.Theconjugationof DTPAwith MAb in a 3.5:1 molar ratio caused the maximum loss of 20% in immunoreactivity of Mab, although the condition gave the MAb

theoptimum labeling fficiency thecombined e in labeling f In o and @Y. TheDTPA-conjugated MAbwaspurified fromunconju

increasentheadministered i dosage of90Y-labeled Ab, asa After the addition of EDTA, the @Y-MAb purified by M was resultof the expected reduction myelotoxicity in (21,22). Sephadex G25 gel filtration. The purified In-MAbnd @Y-MAb a
In this study, the possibility of improvement of RID and R@ITwith In- and 90Y-DTPA-MAb with CaNa2EDTA pre- and post-treatment was investigated, and the efficacy is contained<2% freeradioactive In @Y, and respectively,andthe specific levels of radioactivity were calculated as approximately 74 MBq/mg. For a control, MAb F33-104 was labeled with 125! y a b chloramine-Tmethod(specificactivity, 440 MBq/mg) (24,25).

discussed.
MATERIALS AND METHODS

In VitroInfluenceof Serumon 111ln-MAb @Y-MAb or Cell LinesandAnimalModels

A human colon adenocarcinoma LS 180 cell line expressing carcinoembryonic antigen (CEA) was obtained from the American Type Culture Collection (Rockville, MD) and maintained in Eagle's minimum essential medium with nonessential amino acids in Earle's Balanced Salt Solutions supplemented with 10% fetal bovine serum (FBS). A human pulmonary squamous cell carci noma PC 9 cell line expressing no CEA was cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS. Female nude mice with a BALB/c/nu/nu background were obtained from the Institute of Experimental Animal Research, Gunma University School of Medicine (Gunma, Japan) at 4 wk of age and inoculated subcutaneously in the right rear flank with tumor cells (1 X 10@ cells/mouse). Mice were allowed free access to commercial chow and distilled, deionized water during the experiment.Ambient temperature during the studywasmaintained at 2123C, the mice were exposed to a 12-h light, 12-h dark and @In-MAb @Y-MAb incubated with human normal or was serum or 50 mmol/L PBS, pH 7.5, as a control for 0, 3, 24, 72, 120, and 168 h at 37C an atmosphere of 5% carbon dioxide in air in (10.4 pigMAb/mL). The influence of serum on the radioactivity of radiolabeled MAbs was estimated by cellulose diacetate electropho resis as described (4,24,26). Cellulose diacetate (Separax-S; Fuji

Photo FilmCo.Ltd.,Tokyo, apan) ascutinto110X 10mm J w

strips, soaked in 0.06 mol/L barbital buffer solution, pH 8.6. The strips then were laid between the anode and cathode in a flat bed electrophoresis tank. Each 1-jiL sample was applied to the mid points of the strips using a sample applicator for high-performance liquid chromatography (HPLC). In-acetate,In-DTPA,@Y
acetate,and @Y-DTPA incubated with serum (to approximately 740

kBq/mL) were also subjected to electrophoresis. The radiolabeled chelates were obtained by direct mixtures of chelators and radionu

clides for60mmatroom temperature. Afterelectrophoresis under

constant current of 0.8 mA/strip for 30 rain at room temperature, the strips were dried, cut into smaller 5 X 10 mm strips, and put into plastictubes.Theradioactivityof fractionswascountedwith a cycle. Mean tumor weight was <0.50 g. Animal studies were Nal well scintillation y counter (Aloka, Tokyo, Japan). A 10% performed in compliance with relevant laws relating to the conduct energy window was centered over the 245 keV of In. the @Y For of animal experiments issued by the Gunma University Ethical count, a window was set at 100 keV to infinity, and the counting Committee. efficiency of the Nal well scintillation -ycounter was approximately 12%. After subtraction of levels of radioactivity corresponding to Radiolabeling MAb of free radionuclides and chelated radionuclides, the radioactivity Murine MAb F33-l04 (IgGI) was generated by a conventional remaining at the MAb was calculated as a percentage of the hybridoma method with highly purified CEA from liver metastases radioactivity of the whole strip at each incubation time. The study of colorectalcancerasan immunizing antigenandreactedagainst was repeated 5 times in duplicate. B3 of protein epitopes on the CEA (23,24). MAb F33-l04 (6.67 X on or l0@ mmol) in 2 mL 0.1 mollL NaHCO3 was reacted with In VitroInfluenceof CaNa2EDTA 1111n-MAb @Y-MAb Athymic murine serum was mixed with CaNa2EDTA (Bleian DTPA-anhydride (DOJIN, Kumamoto, Japan) (23.3 X l0@ mmol) in dimethyl sulfoxide at 4C 12 h. These reaction conditions Inj.; Nisshin Pharmacy Co. Ltd., Yamagata, Japan) to a final for gave 1.82.1 DTPAgrOUpS MAb molecule, determined by In concentration of 1.6 mg/mL, corresponding to twice the level of the per

338

THEJouRNAL FNUCLEAR O MEDICINEVol. 41 2 No. February2000

I
initial serum concentration in a biodistribution study described below, or to 0.9% NaCI as a control. In-MAb r @Y-MAb o was then incubated with the mixture at 37C 0, 3, 24, 72, 120, and for Japan) at 3, 24, 72, 120, and 168 h after intravenous injection of
In-MAb or @Y-MAb. The protocol is shown in Figure 1.

168h in an atmosphere 5% carbon of dioxide air (10.4jig in

@ MAb/mL serum). The influence of chelator on the radioactivity of radiolabeled MAbs was estimated by cellulose diacetate electropho resis using the same method as that described above. and @Y-EDTA incubated with serum (to 740 kBq/mL) were also subjected to electrophoresis as additional references.

Circulating blood was replaced by 0.9% NaCl. The right atrium was cut and opened, and, after sampling blood by a heart puncture, 0.9% NaCl was gently perfused into the left ventricle with a small syringe. The tumor and organs were removed and cut into pieces weighing <200 mg, and the volume of the blood sample was <100 jiL. The levels of radioactivity of each piece of tumor, of organs,
and of blood were counted in a Nal well scintillation y counter.

Under this condition, a sufficient linearity between @Y radioactiv In VitroInfluenceof CaNa2EDTA 111ln-MAb on ity and counting rate was obtained. Results were expressed as or mY@MAb Boundto TumorCells percentage injected dose/gram tissue (%ID/g) normalized to a 20-g LS 180 cells were grown to approximately 80% confluence in 4 mouse for each tissue radioactivity. Specific tumor-to-normal tissue dishes (150 X 150 mm) and were preincubated with In-chloride radioactivity ratio was obtained from ([%ID/g LS 180tumor]/[%ID/ (740 kBq) in 20 mL fresh medium supplemented with 10% FBS for g tissue]). Specific tumor (LS 180 tumor)-to-nonspecific tumor (PC 60 mm at 37C an atmosphere of 5% carbon dioxide in air, to 9 tumor) adioactivity in r wascalculated as([mean %ID/gLS 180 avoid the influence of incorporation of radionuclides into tumor tumor]/[mean %ID/g PC 9 tumor]). Serum calcium levels from cells (prelabeling). After triple washing with 20 mL fresh medium, blood of mice with CaNa2EDTAor 0.9% NaCl were monitored at 1 jiL of the mixture of In-MAb serum (10.4 jig MAb/mL) 168h afterinjection theorthocresol and by phthaleinomplexone c incubated at 37 C 60 rain. The cells were then incubated with tumor- or PC 9 tumor-bearing mice at various points during the for 32 mg CaNa2EDTA(l .6 mg/mL) or 0.9% NaCl as a control at 37C study was determined by CEA-immunoradiometric assay kit for 60 mm in air after triple washing with 20 mL fresh medium. Daiichi II (Daiichi Radioisotope Laboratory, Tokyo, Japan). After collection of I X l0@cells in 200 p1, PBS (136.8 mmol/L NaCl, 2.7 mmollL KC1, 8.1 mmol/L Na2HPO4, and 1.5 mmol/L Data Analysis KH2PO4)in microtubes, the cells were separated by centrifugation Statistical comparisons were made by the Student t test. at 10,000g for 5 mm and were counted in a Na! well scintillation @y Differences were considered statistically significant when P < counter. Results were expressed as the mean of [counts/mm 0.05. SD ofcells with CaNa2EDTAJ/[counts/minofcells with 0.9% NaCl] X
100) (%) from a 5-time mnterassay in duplicate. The same proce

incubated for72 or 168h at37Casadded w intothedishesnd method. Circulating CEA concentration in the blood of LS 180 a

dure, except for prelabeling, was performed for @Y-MAb. Cell viability was ascertained at >95% by trypan blue staining. Effect of CaNa2EDTAon Biodlstribution of LS 180

RESULTS In VitroStudies
Loss of radioactivity from In-MAb or @Y-MAb in human serum was approximately 10% and 33%, respec tively, for up to 168 h, whereas loss of radioactivity from In-MAband @Y-MAb PBS was approximately 3% and in
5%, respectively (Fig. 2A). No influence of CaNa2EDTA on

Tumor-or PC9 Tumor-Bearing NudeMicewith

@ 111ln-MAb @Y-MAb or CaNa2EDTA(37.5 mg/kg) was slowly administered intraperito neally before and after intravenous injection of In-MAb r o @Y-MAb jig MAbIkg of mouse body weight; specific activity, (500 74 MBq/mg) to nude mice bearing LS 180 tumor or PC 9 tumor. As a control, 0.9% NaCI was used in place of the chelator. Mice were killed under general anesthesia with diethylether (Wako, Osaka,

loss radioactivityrom In-MAb @Y-MAb of f or wasshown

(Fig. 2B). There was no difference in loss of radioactivity from radiolabeled MAbs between human serum and murine

serum.No effectof CaNa2EDTAn lossof radioactivity o

[n-MAb

injectionor'Y-MAb injection

sacrifice (n=5 at each point)

. .

LS180 Mice
-12-11

24 22 34

,
46

7@2

1@
82 94 1

168(h)

H
PC9 Mice

I
37.5mg/kg CaNa@EDTA-administration or 0.9% NaCI-administration ]

@ @

11 -12-1 1
I
.

3t

I @!

@!

I
12

I
r
at each point)I

1@46@(h) 1

2@
sacrifice

16@(h)

(n=5

[@ln-MAb

injection or90V@MAbinjection

FIGURE Protocol 1. ofinvivo studies.

Ewi@c@r OFEDTA ONRID ANDRIT Watanabe al. et

339

@
@

:
%Radioactivity

@1@

%Radioactivity
. a T . . .. .@ V.. I T .., T A

100@
90.
@ ,@. -.fr ......@ @-

807060. 5040
&

hi@@.. o--In-Ill-MAb inSerum

50 . 40 30 20 . a with0.9%NCI @n-111-MAbinSeruni w1thEDTA
Y.90.MAb m Serum

In@i 11-MAb inPBS

A c V
I I

In-ill-MAb

in Senim

30 20
10

Y-90.MAb in PBS Y-90.MAb in Serum

I I I

ci---

with0.9%Nsa
Y-90-MAb in Serum

10 0@

FIGURE (A)Loss 2. ofradioactMties from

111In-MAb @Y-MAb and in serum.(B) Influ ence of CaNa2EDTA loss of radioactivi on ties from 111In-MAbnd @Y-MAb a in serum. 0

withEDTA

24 48 72 96 120 144 168

24 48 72 96 120 144 168

Time incubation B after (h)

Time incubation after (h)

from In-MAb or @Y-MAb binding to LS 180 cells was shown (99.4% 0.57% for 72-h mixture versus 99.2% 0.39% for 168-h mixture; 98.4% 1.48% versus 98.3% 1.42%, respectively).

In VivoStudies
CaNa2EDTA significantly decreased the %IDIg of tumor in LS 180 tumor-bearing mice from 72 h after injection of In-MAb compared with that of the control (Fig. 3). In addition, the hepatic and renal levels of radioactivity in

CaNa2EDTA-treated mice bearing LS 180 tumor were also significantly reduced compared with those of controls (Fig. 3). With respect to other tissues or blood, CaNa2EDTA had no influence on the radioactive levels (%IDIg). The tumor-to blood ratio in mice bearing LS 180 tumor was significantly decreased from 72 h after injection of In-MAb by CaNa2EDTA compared with that ofthe control (Table 1), but the tumor-to-liver or tumor-to-kidney radioactivity ratios were not affected (Fig. 4). On the other hand, in mice bearing

Blood

(LS180)

uv@ @LS 180)

I
15.
T

5.

120 %IDlg tissue @ 25

168

Time after injectionof In-i 11-MAb(h)

Tumor

(lAS180)
20 15.

(PC 9)

10@TT

I@

@.

FIGURE Effect fpre-andpostadmin 3. o

istered CaNa2EDTAon biodistnbution of
radioactivity in tumor-bearing mice injected
3 24 72 120168 3 ofln-li 24 72 120168

with 111ln-MAb. < 0.05 for CaNa2EDTA treated mice and controls.

Time after injection

1-MAb (h)

340

THE Joui@r@ OF NUCLEAR MEDICINE ol. 41 o. 2 ebruary V N F

2000

TABLE I Effectof Pre- and Postadministered CaNa2EDTA on Tumor-to-Blood Radioactivity Ratio in LS 180 Tumor-BearingMice Injectedwith 1ln-MAb @Y-MAb or Time
after injection1ln-MAb@Y-MAb0.9% NaClCaNa2EDTA0.9% NaClCaNa2EDTA(h)treatmenttreatmenttreatmenttreatment Specific Di@ $0
@P@@4.0

8.0 7.0

is@w@c i 9
...-

6.0 5.o@
I

..... .

0.12242.28 30.35 0.070.36 0.36729.65 0.382.24 4.0512058.2 0.938.49 5.2016889.6 5.3750.6

0.040.69 0.194.44 0.61*36.2 4.90*42.0 6.7579.3 6.60*53.2

0.100.73 6.1542.8 7.4054.6 7.13

0.234.60 5.5237.9

3.0 20
.

fr- In-il1-MAb ith0.9%N@1 w g...- In-lil-MAbwithEDTA

v- Y-90-MAb with 0.9% NaCI

1.0. 0.0. I
24 0

,@. I
48

Y-90-MAb withEDTA I
72

*P < 0.05 between 0.9% NaCI-treated(control) mice and CaNa2EDTA-treated mice. Valuesaremean SD.

96 120 144 168 ofradiolabeled MAbs (h)

Tuue after in@on

FiGURE5. Effectofpre-andpostadministered CaNa2EDTAon specific tumor-to-nonspecific tumor radioactivity ratio of 111In PC 9 tumor, CaNa2EDTA significantly reduced the %ID/g of tumor from 72 h after injection of In-MAb(Fig. 3). The specific tumor-to-nonspecific tumor radioactivity ratio was definitely raised from 72 h after injection of In-MAbby CaNa2EDTA treatment compared with that of the control (Fig. 5). CaNa2EDTA significantly decreased the bony radioactiv ity of mice bearing LS 180 tumor from 24 h after injection of

MAbor @V-MAb.
@Y-MAb compared with that of the control (Fig. 6). The hepatic and renal levels of radioactivity in LS 180 tumor bearing mice were significantly reduced by CaNa2EDTA treatment from 72 h after injection of @Y-MAb, compared with that of the control mice (Fig. 6). CaNa2EDTA had no effect on %ID/g of tumor in LS 180 tumor-bearing mice (Fig. 6). With respect to other tissues or blood, CaNa2EDTA had no influence on the levels of radioactivity (%ID/g). CaNa2EDTA had no effect on tumor-to-blood radioactivity ratio in mice bearing LS 180 (Table 1). CaNa2EDTA significantly raised the tumor-to-bone, tumor-to-liver, and tumor-to-kidney radioactivity ratios in mice bearing LS 180 tumor from 24, 72, and 72 h, respectively, after injection of @Y-MAb, compared with those of controls (Fig. 7). On the other hand, in mice bearing PC 9 tumor, CaNa2EDTA had no effect on the %ID/g of tumor tissue. The specific tumor-to nonspecific tumor radioactivity ratio of @Y-MAb not was influenced by CaNa2EDTA (Fig. 5). No significant growth of the LS 180 or PC 9 tumor was found during these studies. In all mice injected with CaNa2EDTA, no inflammation of the abdominal wall was seen macroscopically. No weight loss or other sign of toxicity such as shivering was observed, nor was any macroscopic change observed in mice kidneys. No signifi cant difference in concentration of serum calcium was noted between the CaNa2EDTA-treated mice and the normal mice (4.62 0.25 mEq/L, n = 39; 4.65 0.15 mEq/L, n = 43). No circulating CEA was detected during the study in the bloodofmicebearingLS l80orPC9tumors(<l ng/mL).

In-lil-MAb (15S180) 2.0

Tumor-to
liver, kidneys

1.5.

1.@
@---@ A Liver(0.9% Liver (EDTA) NaC1)

u----- Kidneys(0.9%NaC1)
,.. Kidneys (EDTA)

0.5.

24

48

72

96

120 144 168

Time after injection (h) FiGURE4. Effectof pre-andpostadministered CaNa2EDTA on tumor-to-liver and -kidney radioactivity ratios in LS 180 tumor
bearing mice injected with @1ln-MAb.

DISCUSSION
CaNa2EDTA did not deplete the levels of radioactivity of radiolabeled MAbs in vitro. In the tumor-bearing mice, CaNa2EDTA pre- and post-treatment reduced the radioactiv ity in both specific and nonspecific tumors at 72 h after

EmicrropEDTAONRID @r@ir PIT Watanabe al. et

341

%ID/g tissue 25Tumor

(LS180) (LS180) El 0.9% NaC1-treatment

CaNa@EDTA-treatment

Blood

Liver

(LS180)

TT T
IT
jj@IT T*

@!.L1IL.
I I@ --@

3
%ID/g tissue

24

72 120168

iiDfl:Li
i

24

72

120 168

Time after injection of Y-90-MAb (h)

2520@

Kidneys

(LS180)

Bone

(LS180)

15 -

10-

T TT
I

TI
* *

5-

I'

r]1@rnI'rThn@LE}@
3 24 72 120168 3 24 72 120168
Time after injection of Y-90-MAb (h)

1@i@T*@*

FIGURE6. Effectofpre-andpostadministered CaNa2EDTAon biodistribution radioactivity of inLS l8otumor-beanng miceinjected with @Y-MAb. 0.05 forCaNa2EDTA-treated iceandcontrols. < m In-MAb-injection, and it raised the specific tumor-to radioactivity in either specific or nonspecific tumors, nor did it influence the specific tumor-to-nonspecific tumor radioac nonspecific tumor radioactivity ratio but lowered the spe cific tumor-to-blood radioactivity ratio. In the LS 180 tivity ratio or the tumor-to-blood radioactivity ratio. Pre- and tumor-bearing mice, pre- and postadministration of postadministered CaNa2EDTA reduced the bony, hepatic, CaNa2EDTA decreased the hepatic and renal levels of and renal levels of radioactivity in the LS 180 tumor-bearing radioactivity at 72 h after In-MAb injection but did not mice at 24, 72, and 72 h after @Y-MAb injection and raised affect the tumor-to-liver or -kidney radioactivity ratios. By the specific tumor-to-bone, -liver, and -kidney radioactivity contrast, in the tumor-bearing mice injected with @Y-MAb,ratios. When not used in combination with preadministra pre-and post-treatment with CaNa2EDTA did not affect the ton, postadministration only of CaNa2EDTA had no effect

342

THE Joui@L

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MEDICINE ol. 41 o. 2 ebruaiy V N F

2000

Tumor-to liver, kidneys 2.5

ratio

Tumor-to

10
ratio

24

48

72

96

120 144 168

Time after injection (h)

FIGURE7. Effectof pre-andpostadmin isteredCaNa2EDTA tumor-to-liver, on -kid ney,and-boneradioactMty ratiosinLS 180 tumor-bearing mice injected with @Y-MAb. *P < 0.O5forCaNa2EDTA-treated miceand

controls.
matory tissue (30). In activity was rarely seen in necrotic tumor tissue. Therefore, it was reasonable to assume that the free @In disassociated from In-MAb by catabolism or instability of the labeling (or both) or internalization was retained in the tumor, partly in the tumor cells and partly in the extracellular space. Furthermore, CaNa2EDTA treatment chelated the free, extracellular In(and the CaNa2EDTA postadministration of CaNa2EDTA was shown to more might inhibit the incorporation of In the tumor cells), into powerfully control the levels of radioactivity in the tumor resulting in reduced radioactivity in the tumor. The finding and bone, liver, and kidneys, respectively, than postadmiis that free radioactivity from In-labeled MAb may be tration only (unpublished data, Watanabe N, December retained in tumors was reported in the early stage of an 1998). Pretreatment with CaNa2EDTA also appears to be investigation into RID (3133). he free radionucide disso T viable for the immediate removal of extracellular free ciated from @Y-MAb a strong potential for redistribution has radionucides. into bone. In a previous study we showed that CaNa2EDTA Soluble Inmay localize in human malignant tumors could not remove the @Y binding to the bone (unpublished without preference for cell type (27,28). The site of In data, Watanabe N, December 1998). Therefore, this accumulation in tumor cells was the cytoplasmic lysosomes (29). Tumors include extracellular components such as CaNa2EDTA-mediated effect on bony radioactivity was the liberated from the radiolabeled connective tissue and vessels, in addition to cells, and result of chelation of the @Y MAb before localization in bone. The early distribution of various pathologic changes such as necrosis and inflamma radioactivity in the liver and kidneys appeared to be the tion. In tumors xenografted in nude mice, the actual result of radiolabeled MAb itself binding to cells, through distribution of Inwas predominant in connective tissue the Fc receptor in most cases. Sequentially, free radionu (especially inflammatory tissue) rather than in viable tumor clides are a result of the catabolism and inherent instability cells themselves (30). The In bound to acid mucopoly was of radiolabeled MAbs. Finally, both radiolabeled MAbs and saccharides or the sulfated carbohydrate chain of sulfated glycoprotein produced as intercellular substances in inflam freed radionuclides are nonspecifically distributed in the on raising the specific tumor-to-nonspecific tumor radioactiv ity ratio in In-MAb, and the power of reduction in bony, hepatic, and renal levels of radioactivity of @Y-MAb was weakened (data not shown). The introduction of pretreat ment with CaNa2EDTA showed an early effect on bony radioactivity for @Y-MAb. tumor-bearing mice with In In-chloride and normal mice with @Y-acetate, and pre-

E@cr

OF EDTA ON RID

@r'@r atanabe RIT W

et a!.

343

14. Quadri SM, Shao Y. B1umJE, Leichner PK, Williams JR. VriesendorpHM. liver and kidneys. It is assumed that CaNa2EDTA scavenges Preclinical evaluation of intravenously administered @1n- @Y-labe1ed and B72.3 the free, extracellular radionucides in the liver and kidneys. immunoconjugate (GYK-DTPA) in beagle dogs. Nuci Med BIOL 1993;20:559Thus, pre- and post-treatment with CaNa2EDTA has an 570. 15. Mardirossian G, Wu C. Hnatowich DJ. The stability in liver homogenates of effect on the improvement of specificity of tumor radioactiv indium-ill and yttrium-90 attached to antibody via two popular chelators. Nuci ity in RID, in addition to reducing hepatic and renal levels of MedBioI. 1993;20:6574. radioactivity (improvement of imaging quality for diagno 16. KosmasC. Snook D. Gooden CS, Ct al. Developmentof humoral immune responses against a macmcycic chelating agent (DOTA) in cancer patients sis). On the other hand, pre- and postadministered receiving radioimmunoconjugates for imaging and therapy. Cancer Res. 1992;52: CaNa2EDTA may permit a sufficient increase of @Y-MAb 904911. dosage for cancer treatment in RIT, because of the increase 17. VriesendorpHM. Herpst JM, Leichner PK, Klein JL. Order SE. Polyclonal 90yttrium-labeled antifenitin for refractory Hodgkin's disease. ln:JRadia: Oncol in the tumor-to-bone, tumor-to-liver, and tumor-to-kidney BloiPhys. 1989;17:815821. radioactivity ratios. 18. KlaassenCD. Toxicology. In: GoodmanLS, Oilman AG. Rail 1W, Nies AS.
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