[CANCER RESEARCH 47, 5427-5432, October 15, 1987]

Radiolocalization of Xenografted Human Lung Cancer with Monoclonal Antibody 8in Nude Mice1

Katsuyuki Endo,2 Hiroshi Kamma, and Takesaburo Ogata

Department of Surgery, Institute of Clinical Medicine ¡K.E.J, Pathology Division, Tsukuba University Hospital [H. K.J, and Department of Pathology, Institute of BasicMedical Sciences fT. O.J, University of Tsukuba, Sakura-mura, Niihari-gun, Ibaraki-ken 305, Japan

ABSTRACT

A monoclonal antibody (MAb) 8 (¡mmunoglobulin G3 (IgG3)|, directedagainst a M, 48,000 human lung cancer-associated antigen, was radiola-beled with either 12!I or '"I, and its biodistribution was studied in nudemice bearing human lung cancer (TKB-2) over a 7-day period. I25l-labeled

MAb 8 increased its binding to the tumor during the period, while thebinding of 125I-labeled control IgG3 declined after initial uptake. At Day7, percentages of injected dose of I25l-labeled MAb 8 bound to the tumor

rose to 7.4%, which was a 4.4-fold increase from Day 1 and 16-foldbinding of I25l-labeled control IgG3 at the same day. Tumonblood ratios

became 2.7:1 at Day 7, and tumonliver, tumonspleen, and tumorkidneyratios were more than 9:1. Normal organs showed no significant uptakeof I25l-labeled MAb 8, compared with those of '"¡-labeled control IgG3.

A clear image of the xenografted tumor was obtained at Day 5, and itfurther improved at Day 7, when 60% of whole body radioactivity waslocalized to the tumor. Autoradiography of the mouse with tumor confirmed the excellent localization of 12il-labeled MAb 8 to the tumor,

although the radioactivity of the tumor was not uniformly distributed.Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis

showed that most of the radioactivity was present at the tumor in theform of degraded immunoglobulin. MAb 8 has a potential usefulness inthe diagnosis and treatment of lung cancer.

INTRODUCTION

The use of specific antibodies for the detection and treatmentof malignancy has been suggested. Early attempts at tumorimaging showed limited success (1-3). The major difficulty hasbeen the production of antibodies that react only with neoplasticcells. The recent development of monoclonal antibodies againsttumor-associated antigens gives promise to such an approach(4).

We developed a number of monoclonal antibodies againsthuman lung cancer. One of these antibodies, MAb3 8, wasproduced using TKB-2, a morphological variant cell line ofhuman small cell carcinoma of the lung, as the immunogen (5).It was IgG3k in isotype, and recognized an M, 48,000 glyco-protein. MAb 8 reacted with 87% of large cell carcinoma, 82%of both squamous cell carcinoma and adenocarcinoma, and19% of small cell carcinoma of the lung by immunoperoxidasestaining. The overall reactivity with lung cancer was 68%. Itbound to the cytoplasm as well as the cell membrane of thecancer cells. Cross-reaction with normal tissues was limited tothe fetal and adult lung, in which the bronchial and alveolarepithelium were slightly stained.

As preliminaries for clinical trials, a detailed assessment ofthe biodistribution and pharmacokinetics of the antibody is

Received 11/5/86; revised 6/29/87; accepted 7/23/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This study was supported in part by a grant from the Japanese Ministry ofEducation. Science and Culture (No. 61771005).

2To whom requests for reprints should be addressed.3The abbreviations used are: MAb 8, a monoclonal antibody directed to a M,

48,000 lung cancer-associated antigen; TKB-2, a morphological variant cell lineof human small cell carcinoma of the lung; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; %ID/g, percentage of injected dose per gramof tissue, corrected for physical decay.

important. In this study, we radioiodinated MAb 8, and examined its localization in tumor-bearing nude mice.

MATERIALS AND METHODS

Monoclonal Antibodies. The monoclonal antibody, MAb 8, was produced by fusion between mouse myeloma cells (P3UX63Ag8Ui) andmouse spleen cells immunized with TKB-2 cells, a morphologicalvariant line of human small cell carcinoma of the lung (5). Its isotypewas IgG3k. As a control, another monoclonal antibody of the sameisotype directed to an irrelevant antigen was used. The antibodies werepurified from ascitic fluid of mice by saturated ammonium sulfateprecipitation and DEAE-Sephacel (Pharmacia Fine Chemicals, Uppsala, Sweden) chromatography. The immunoglobulin concentration wasdetermined by absorbance at A..«„.

Radiolabeling of Monoclonal Antibodies. Purified MAb 8 and thecontrol IgG3 were radiolabeled with either I25I or I3II (AmershamCorporation, Arlington Heights, IL) using the IODO-GEN (PearceChemical Company, Rockford, IL) method. Briefly, 100 u% of theantibody were mixed with 1 to 2 mCi of the radioisotope in a tubecoated with 100 ^g of IODO-GEN for 10 min on ice. The reaction wasstopped by adding 1 HIMt>rosine, and free isotope was separated witha Sephadex G-25 column (Quik-Sep QS-2A; Isolab, Inc., Akron, OH).Specific activity of the radiolabeled antibodies ranged from 1 to 10uCi/ug. Immunoreactivity of the radioiodinated antibody preparationwas evaluated by enzyme-linked immunosorbent assay (S) using thenonlabeled antibody as a control.

In Vivo Localization. BALB/c female nude mice 4 to S weeks oldwere inoculated 1 x 10' s.c. in the flank with TKB-2 cells. The tumor

grew to 1.0 to 2.0 cm in diameter by 4 to 6 weeks. The mice wereadministered i.p. 4 to 10 uCi of either '"I-labeled MAb 8 or '"I-labeledcontrol IgG3. They were sacrificed successively over a 7-day period byexsanguination, and radioactivity of the tumor and every organ (cpmper mg) was counted. Percentage of injected dose per gram of tissue,corrected for physical decay, was calculated. Tumontissue ratio wasdetermined from this data. The thyroid was blocked with 0.1% KI indrinking water, starting 5 days before injection.

Radioimaging. The TKB-2 tumor-bearing mice were given i.v. 80 //Ciof either ml-labeled MAb 8 or 13ll-labeled control IgG3. Scanning was

performed using a gamma camera (Photogamma LFOV; Shimazu-Searle, Inc., Kyoto, Japan) equipped with a pinhole collimeter of 8 mmin aperture. A 20% energy window was centered over 364 keV. Datawere stored on a computer (Shimazu Scintipac 230; Shimazu, Inc.,Kyoto, Japan). The region of interest with regard to the tumor wasapplied to the recorded image, and percentage of whole body radioactivity in the tumor was determined. Background subtraction was notutilized.

Autoradiography. The mice with TKB-2 tumor were given i.p. 4 uCiof 125I-labeled MAb 8. One mouse was sacrificed by dipping it into

liquid nitrogen at Day 5. A sagittal whole-body section of SO urn inthickness was cut at the left kidney plane. Autoradiography was performed using Fuji RX film (Fuji Photo Film Company, Ltd., Tokyo,Japan). In another mouse, the tumor was excised on Day 7, fixed withformalin, and embedded in paraffin. Both \5-um sections kept incontact with Fuji RX film and S-^m sections coated with Sakuraautoradiographic emulsion NR-M2 (Konishiroku Photo Ind. Corporation, Ltd., Tokyo, Japan) were exposed for 2 months.

SDS-PAGE. A mouse bearing TKB-2 tumor was given an injectioni.p. of 20 /iCi of 125I-labeledMAb 8, and sacrificed at Day 7. The tumorwas minced, homogenized in Ca2*and Mg2+free Dulbecco's phosphate-

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LOCALIZATION OF XENOGRAFTED LUNG CANCER WITH MAb 8

Table 1 Distribution of "¡-labeled monoclonal antibodies in nude mice with TKB-2 tumor

Nude mice were inoculated s.c. in the flank with TKB-2 cells, human small cell carcinoma of the lung. Mice with a tumor of 1.0 to 2.0 cm in diameter wereadministered i.p. 4 to 10 /jCi of either '"I-labeled MAb 8 or '"I-labeled control IgG3 and sacrificed over a 7-day period. The radioactivity (cpm per mg) of the tumor

and various organs was counted and corrected for physical decay. The percentage of the injected dose per gram of tissue was calculated from this data; results are themean ±SD of 3 to 5 mice.

Organ

% of injected dose/g

Day 1 (n = 3) Day 3 (n = 3) Day 5 (n = 3) Day 7 (n = 5)

'"I-labeled MAb8TumorBloodBrainThyroidLungStomachLiverSpleenKidneyColonBoneMuscleSkin'"I-labeled

controlIgG3TumorBloodBrainThyroidLungStomachLiverSpleenKidneyColonBoneMuscleSkin1.66

±0.128.86±2.450.24±0.023.09±0.484.99±0.23.78+0.36.90±0.23.90

±0.122.97±0.35.78±0.37.24

±0.13.06±0.384.39±0.491.66

±0.209.77±2.410.20±0.022.54

±0.105.37±0.291.76

±0.491.76±0.032.15±0.112.54

±0.121.47±0.291.17±0.210.88

+0.124.56±0.683.65

±1.156.61±2.610.25

±0.082.32±0.253.07±0.09.25±0.34.33±0.33.08±0.02.99±0.42.16

±0.340.91±0.180.99

±0.262.82+0.512.02

±0.566.98±4.030.21

±0.022.02±0.353.21

+0.36.12±0.14.40

±0.21.68±0.84.54±0.07.05±0.03.05±0.300.84±0.082.58±0.225.70

±0.944.24+1.840.17±0.061.48±0.172.33

±0.230.91±0.111.14±0.070.94

+0.121.54+0.290.80±0.180.63+0.070.57±0.062.16+0.971.87

±0.175.59+3.050.22±0.061.45+0.112.96±0.070.84±0.061.17

±0.121.29±0.181.23±0.060.84

±0.050.89+0.060.67+0.062.24±0.117.37

±1.452.78±0.790.15+0.061.12+0.071.42

±0.100.85±0.420.66±0.060.62±0.160.81

±0.110.70±0.170.50±0.160.50

±0.101.66+0.180.47

±0.182.55+1.130.11±0.041.01+0.181.45±0.210.64±0.110.60

±0.050.60±0.040.84±0.070.58+0.040.49±0.040.41

±0.121.40±0.05

1.0

08

06

I

0«

02

BLOOD LIVER SPLEEN LUNG KIDNEY

t I. 16 64 256 1024 40% 16384DILUTION ( RECIPROCAL)

Fig. 1. Enzyme-linked immunosorbent assay of ''1 labeled and nonlabeled

MAb 8 to TKB-2 cells. The assays were performed using serial 4-fold dilutionsof '"I-labeled (O) and nonlabeled (•)MAb 8 starting with 4 jig/ml. Results arethe mean of quadruplicate assays.

buffered saline with 0.5% Nonidet P-40 and 1 mM phenylmethylsulfonylfluoride on ice, and centrifuged at 10,000 x g for 10 min at4°C.'"I-labeled MAb 8, supernatant of the homogenized tumor, andthe mouse serum were subjected to SDS-PAGE, and autoradiographywas performed. As a control, 12SI-labeled MAb 8 was added to theexcised tumor from a mouse not given an injection of I25l-labeled MAb8, and the sample was processed in parallel. Low-molecular-weightprotein standard (Bio-Rad Laboratories, Richmond, CA) was used as amolecular weight marker.

RESULTSImmunoreactivity of I25l-labeled MAb 8. Immunoreactivity of

125I-labeled MAb 8 was well maintained after iodination. En-

12

DIO

luoí/)6

04

1357 1357 1357DAYS AFTER INJECTION

Fig. 2. Distribution of '"I-labeled antibodies in nude mice with TKB-2 tumor.Mice with TKB-2 tumor were given injections i.p. of 4 to 10 /iCi of either '"I-labeled MAb 8 (O) or '"I-labeled control IgG3 (•),and sacrificed successively.

The radioactivity of tissue (cpm per mg) was counted, and tumontissue ratio wascalculated. Results are the mean of 3 to 5 mice.

zyme-linked immunosorbent assay demonstrated the similarbinding of the labeled and non-labeled MAb 8 to TKB-2 targetcells (Fig. 1).

In Vivo Distribution. The TKB-2 tumor-bearing mice wereadministered i.p. 4 to 10 ¡iC\of either I25l-labeled MAb 8 orI25l-labeled control IgG3 and sacrificed over a 7-day period.After '"I-labeled MAb 8 injection, percentages of injected dose

bound to the tumor (Table 1) increased progressively throughout the period, while those after 125I-labeled control IgG3

injection decreased after initial uptake during the first 3 days.At Day 7, the tumor took up 7.37% of 125I-labeled MAb 8injected, which was a 4.4-fold increase from Day 1 and a 16-fold binding of I25l-labeled control IgG3 on the same day.Normal organs examined showed no significant uptake of 125I-1abe led MAb 8, compared with those of I25l-labeled control

IgG3. Tumontissue ratios also rose over this period (Fig. 2).5428

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LOCALIZATION OF XENOGRAFTED LUNG CANCER WITH MAb 8

Fig. 3. Scintigraphic image. I3ll-labeledMAb 8, 80 /.< i. was injected i.v. into a nudemouse bearing TKB-2 tumor (a), and scanningwas performed at Days 3 (b), 5 (c), and 7 (if).Data were stored on a computer, and countingtime was adjusted to 100 s. Background snli-traction was not utilized.

Table 2 Distribution of " ¡-labeledmonoclonal antibodies in nude micewith TKB-2 tumor

Mice with TKB-2 tumor were given an i.v. injection of 80 ¿iCiof either '"I-labeled MAb 8 or '"I-labeled control IgG3. Gamma camera scanning wasperformed successively, and the region of interest in regard to the tumor wasmeasured; results are the mean of 3 mice.

Daysafter

injection1

357'"I-labeled

MAb(n =3)%

total inmouse"100

68.420.012.6%

total intumor*17.1

22.450.259.8'"I-labeled

control IgG3(n =3)%

total inmouse"100

62.822.518.3%

total intumor*15.3

13.811.910.5

" Percentage of total radioactivity of Day 1 that remained in the mouse.* Percentage of total radioactivity of the mouse found in the tumor.

Tumorblood ratio became 2.7:1 at Day 7. Tumor.liver, tu-

monspleen, and tumonkidney ratios increased to more than9:1, while those with '"I-labeled control IgG3 remained less

than 2:1.Imaging of Tumor-bearing Mice. The mice bearing the 1KB

2 tumor were given injections i.v. of 80 ßdof either '"I-labeledMAb 8 or I3'l-labeled control IgG3, and studied by gamma

camera scanning (Fig. 3). Up to Day 3, the whole body of themice was imaged and the tumor border was difficult to define.The tumor image became increasingly clear as body backgroundsubsided and was distinct at Day 5. At Day 7, most of theradioactivity was seen at the site of the tumor. The backgroundactivity cleared with a i./,of approximately 8.7 days. As for 13II-

labeled control IgG3, tumor image was not apparent throughoutthe study. Percentages of whole body radioactivity in the tumor(Table 2) increased from 17.0% at Day 1 to 59.8% at Day 7,while those of control IgG3 remained at less than 20%.

Autoradiography of Mice. The mouse xenografted with 1KB2 tumor was sacrificed 5 days after 125I-labeledMAb 8 injection,

and autoradiography of the whole body section was performed(Fig. 4). Although radioactivity was observed throughout thebody including liver, kidney, and lung, the tumor showed thestrongest uptake of 125I-labeled MAb 8. Distribution of radioi-

sotope at the tumor was not uniform, some areas showingstrong binding and others remaining unbound. Except for thetumor, the blood vessels around the eye and those at the neckcontained moderate radioactivity. Slight activity at the skin wasdue to contamination with urine and fèces.As demonstrated inFig. 5, the central necrotic area of the tumor showed no radio-

Fig. 4. Autoradiography of a whole bodysection of a mouse with TKB-2 tumor. Amouse was sacrificed 5 days after injecting i.p.4 >iCi of '"I-labeled MAb 8. a, autoradiogra

phy of the whole body section; b, its schema./. tumor; 2, kidney: '. spleen; 4, stomach: 5,liver; 6, lung: 7, inguinal skin.

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LOCALIZATION OF XENOGRAFTED LUNG CANCER WITH MAb 8

Fig. 5. Autoradiography of paraffin-embedded tumor sections. A mouse with TKB-2tumor was sacrificed 7 days after injecting i.p.4 jiCi of '"I-labeled MAb 8. a, hematoxylinand eosin stain: ft. autoradiography; c, microautoradiography (X 100) of the tumor. Vnecrotic area; V, viable area.

k92.5-66.2-

45-

31-

21.5-

Fig. 6. Autoradiography of SDS-PAGE analysis of '"I-labeled MAb 8, tumorhomogenate. and mouse serum. A TKB-2 tumor-bearing mouse was given aninjection i.p. of 20 jiCi of '"I-labeled MAb 8 and sacrificed at Day 7. As a control.'"I-labeled MAb 8 was added to the excised TKB-2 tumor and serum from amouse without I25l-labeled MAb 8 injection, and the samples were processed in

parallel. Lanes 1 to 5, nonreduced condition, and Lanes 6 to 10, reduced condition.The injected '"I-labeled MAb 8 (Lanes I and 6), supernatant of TKB-2 tumorhomogenate (Lanes 2 and 7), mouse serum (Lanes 3 and 8), control tumorhomogenate (Lanes 4 and 9). and control serum (Lanes 5 and 10) were analyzedby 10% SDS-PAGE. The amounts of the samples were adjusted to similar cpm.A.thousands.

activity, but the surrounding viable area took up 125I-labeled

MAb 8.SDS-PAGE Analysis. SDS-PAGE analysis (Fig. 6) of the

radioiodinated MAb 8 showed very strong radioactivity by theheavy chain compared with that of the light chain. In the tumorhomogenate, most of its radioactivity was present at F(ab')2

and the heavy chain, and only a trace amount at IgG. In thecontrol tumor, on the other hand, most of the radioactivity waspresent at IgG. Both of the mouse sera showed I25l-labeled IgG.

DISCUSSION

Monoclonal antibodies conjugated to radioisotopes have beenstudied for localization of malignancy by a number of investigators. In experimental animals, antibodies directed to carci-noembryonic antigen (6-10), melanoma (11, 12), colon carcinoma (13-15), breast carcinoma (16), hepatocarcinoma (17),osteogenic sarcoma (18, 19), renal cell carcinoma (20), germcell tumor (21), and milk fat globule (22) have been used. Inclinical studies, melanoma (23-25), colon carcinoma (26, 27),and breast carcinoma (28, 29) have been their targets. Most ofthese antibodies were highly specific to tumor-associated antigens, but some antibodies detected tumors in spite of thepresence of cross-reacting normal tissues (30, 31). Not only

8 9 10 intact IgG, F(ab'h and Fab fragments were also used successfully (7, 8, 13, 16, 23, 25-27). 13II, '"I, I23I (28), and '"In (9,

10, 17, 22, 24, 29) were used for labeling in most cases.The present study demonstrated that radioiodinated MAb 8

had accumulated at the tumor xenografted in nude mice andhad given a distinct image of the tumor by scanning. Thespecificity of this localization to the tumor was supported bythe absence of any significant accumulation of ' '^1-labeled con

trol IgG of the same isotype. The increasing tu moni issue ratiosover the period studied and the predominant radioactivity atthe tumor by autoradiography also confirmed the specificity ofradioiodinated MAb 8 for the tumor.

The immunoreactivity of 125I-labeled MAb 8, which had beenlabeled by the IODO-GEN method, was well maintained. SDS-PAGE analysis showed an intensive radioactivity by heavy chainand an only slight incorporation of I25I by light chain, even

taking into account the difference in molecular weight betweenthe two. Probably the light chain of MAb 8 contains a fewexposed tyrosine residues in its chain. Although dehalogenationis one of the serious problems in studying radioiodinated antibodies in vivo (32, 33), radioiodinated MAb 8 was stable enoughto give a clear image of the tumor in this study.

Several parameters have been used to indicate the localizationof radiolabeled antibodies. Tumorblood ratios increased progressively after 125I-labeled MAb 8 injection and reached 2.7:1

at Day 7, while those of the control IgG remained at less than0.6:1 during the period. This increase of tumonblood ratios wasin part due to the clearance of the radiolabeled antibody fromthe blood but was also due to the continuous accumulation ofI25I-labeled MAb 8 by the tumor for as long as 7 days. Tu-

monblood ratio may be influenced by the amount of antibodyinjected and the size of tumor, and %ID/g is another parameter(19). %ID/g of the tumor continued to increase over the period,indicating that the accumulation of 125I-labeled MAb 8 by the

tumor still lasted even after 7 days. A third parameter, percentage of whole body radioactivity in the tumor, also continued torise during the period. This continuous accumulation of antibody in the tumor was different from other reports (9, 13, 15,19) in which %ID/g of the tumor had reached maximum 2 to3 days after antibody injection and had decreased slowly thereafter. This long accumulation and retention of MAb 8 in thetumor is particularly desirable for a therapeutic application,since the prolonged contact of drug- or isotope-conjugatedantibody with tumor cells enables effective killing of them.

Interestingly, SDS-PAGE analysis of the tumor homogenateshowed that most of the injected radioactivity was present atthe tumor in the form of 125I-labeled F(ab')2 and heavy chain,and I25l-labeled intact IgG was present in only a trace amount.

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LOCALIZATION OF XENOGRAFTED LUNG CANCER WITH MAb 8

There are two possibilities to explain the degradation of administered '"I-labeled MAb 8 in the tumor. One possibility is that

the injected MAb 8 was decomposed after homogenization withendogenous proteolytic enzymes released from broken cells.Another possibility is that MAb 8 was degraded /// situ in thetumor before excision. Although the former possibility cannotbe excluded, the latter is more likely: (a) the control sample, inwhich I25I-labeled MAb 8 was added after excision, showed

only slight degradation; (b) MAb 8 reacted strongly with thecytoplasmic antigen by immunoperoxidase staining (5). It maybe acceptable to think that I25l-labeled MAb 8 was internalized

to bind the cytoplasmic antigen and degraded in the cytoplasm.The slow and continuous accumulation of '"I-labeled MAb 8

by the tumor may be related to the internalization of MAb 8into TKB-2 tumor cells in situ.

The heterogeneous nature of malignant tumor is one of thedifficult problems to be resolved for the detection and treatmentof malignancy with monoclonal antibodies. As revealed byautoradiography of the whole body section of a mouse, radioactivity at the tumor was not uniform. This inhomogeneity waslargely due to the presence of necrosis in the tumor. Most ofthe viable regions reacted with I25l-labeled MAb 8, as shown by

microautoradiography. However, this may not be the case inclinical situations, since immunoperoxidase staining with MAb8 showed heterogeneous distribution of the antigen within alung cancer (5). Two suggested resolutions for this difficultyare the administration of a group of monoclonal antibodieswith different binding repertories (4) and the coupling with anappropriate radioisotope that can kill several cell diameters inthe hope that one cell, at least, in a tumor cluster expresses theantigen (15). Further studies are necessary to settle the problem.

In comparison with I25l-labeled control IgG3, I25l-labeled

MAb 8 showed no significant binding to normal organs. Theslight cross-reaction, which had been demonstrated with thehuman lung by immunoperoxidase staining (5), was not significant in this study. The discrepancy may originate in the difference of species or method of study and indicates that great caremust be taken in applying to clinical cases the results of a studyusing mice (34-36).

In summary, radioiodinated MAb 8 localized well to thetumor xenografted in nude mice and gave an excellent imageof the tumor. The continuous accumulation by the tumor ofradioiodinated MAb 8 is desirable for therapeutic application.However, great care must be taken in interpreting these datafor clinical trials.

ACKNOWLEDGMENTS

The authors wish to thank R. Hatakeyama for his technical assistancein scanning, Y. Hattori for his assistance in autoradiography, andProfessor M. Akisada and Dr. N. Ishikawa, Department of Radiology,for their suggestions.

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1987;47:5427-5432. Cancer Res   Katsuyuki Endo, Hiroshi Kamma and Takesaburo Ogata  Monoclonal Antibody 8 in Nude MiceRadiolocalization of Xenografted Human Lung Cancer with

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