In-labeled Monoclonal Antibody 96.5 111 ...

Radioimmunoimaging in Malignant Melanoma with 111 In-labeled Monoclonal Antibody 96.5 James L. Murray, Michael G. Rosenblum, Robert E. Sobol, et al. Cancer Res 1985;45:2376-2381. Published online May 1, 1985.

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[CANCER RESEARCH 45, 2376-2381.

May 1985]

Radioimmunoimaging in Malignant Melanoma with 111ln-labeled Monoclonal Antibody 96.51 James L. Murray,2 Michael G. Rosenblum, Robert E. Sobol, Richard M. Bartholomew, Carl E. Plager, Thomas P. Haynie, Monroe F. Jahns, Howard J. Glenn, Lamk Lamki, Robert S. Benjamin, Nicholas Papadopoulos, Arthur W. Boddie, James M. Frincke, Gary S. David, Dennis J. Carlo, and Evan M. Hersh M. D. Anderson Hospital and Tumor Institute, the University of Texas System Cancer Center, Houston, Texas 77030 [J. L. M., M. G. R., C. E. P., T. P. H., M. F. J., H. J. G., L L, R. S. B., N. P., A. W. B., E. M. H.J; University of California, San Diego Cancer Center and Veteran's Administration Medical Center, San Diego, California 92161 [R. E. S.]; and Hybritech, Inc., San Diego, California 92121 [R. M. B., J. M. F., G. S. D., D. J. C.]

ABSTRACT A radiolabeled monoclonal antibody (96.5) reactive with an M, 97,000 antigen found on over 80% of melanoma cell lines and tissue extracts was examined for its ability to detect malignant melanoma mÃ©tastases in vivo. For imaging purposes, it was conjugated with diethyltriaminepentaacetic acid and subse quently labeled with 111lnby chelation. Thirty-one patients with metastatic melanoma received single injections of monoclonal antibody 96.5 at concentrations ranging from 0.5 to 20 mg and at specific activities of 111lnranging from 0.125 to 4 mCi/mg. Total-body scans were performed at various time intervals fol lowing administration. No serious side effects were observed. Of a total of 100 previously documented metastatic sites, 50 imaged for a specificity of 50%. The number of sites imaged increased significantly as the amount of antibody administered increased relative to the average radiation dose. Considerable background uptake of isotope was observed in blood pool and other organs with gradual acquisition of label in tumor sites by 48 to 72 h. Hence, tumor imaging of melanoma using 111In-labeled monoclo nal antibody 96.5 appeared feasible, especially at antibody doses above 2 mg.

for developing large quantities of tumor-specific MoAb for use in immunodiagnosis and therapy of cancer. In addition, the use of alternate radionuclides to iodine, such as 111ln, has minimized the problem of in vivo dehalogenation (8). On the other hand, the ratio of tumor to nontumor uptake of 111lndid not appear to offer significant advantages over 131Ias confirmed in several animal models (7, 21). In this paper, we have examined the safety as well as efficacy of radioimmunoimaging using 96.5, a MoAb reactive against human malignant melanoma. The melanoma-associated antigen p97 is found on over 80% of melanoma cell lines and tumor extracts (23). It is also expressed in low concentration on breast tumor lines, normal melanocytes, and fetal tissues (3, 22), and it is functionally related to transferrin (2). Antibody 96.5 is of the lgG2a subclass and is cytotoxic to melanoma cell lines in the presence of rabbit complement in vitro (9). The antibody, covalently coupled to the chelating agent DTPA, was provided with the 111lnto be added at the time of the study by Hybritech, Inc., San Diego, CA. The pharmacokinetics detailed in a companion paper (18).

Preparation of "'In-labeled

and human chorionic gonadotropin, to image human tumors in vivo (5, 6, 10). The majority of these studies used 131Ias the radionuclide. Although successful visualization of tumors was reported, the low ratios of tumor to normal tissue uptake of labeled antibody often necessitated the use of background sub traction techniques using a second antibody or albumin (6). The advent of hybridoma technology has created the potential ' This work was supported by a grant from Hybritech, Inc. 2 To whom requests for reprints should be addressed, at Clinical Immunology, Box 41, M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Avenue, Houston, TX 77030. 'The abbreviations used are: CEA, carcinoembryonic antigen; MoAb, monoclo nal antibody(ies); p97, glycoprotein with a molecular weight of 97,000; DTPA, diethyltriaminepentaacetic acid; SGOT, serum glutamic-oxaloacetic transaminase; BUN, blood urea nitrogen; i.d., intradermally. Received 5/3/84; revised 10/1/84,1/18/85; accepted 1/25/85.

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is

MATERIALS AND METHODS

INTRODUCTION In 1948, Pressman and Keighley (16) demonstrated that polyclonal antibodies to rat kidney which had been coupled to 131Icould be used to image the appropriate renal tissue. Addi tional studies using dual isotopes and tissue-counting techniques (15) confirmed the specificity of antiserum for kidney tissue. Since this important discovery, other investigators have exam ined the feasibility of using radiolabeled polyclonal antibodies reactive with oncofetal antigens, including CEA,3 a-fetoprotein,

of this preparation

96.5. The MoAb to p97 was made by

immunizing BALB/c mice with a melanoma cell line (SK-Mel-28) and fusing their splenocytes with the NS-1 myeloma cell line to form hybrids as has been described previously (24). The hybridoma cells were grown in BALB/c mice as ascites, and the antibody was purified from ascites fluid using DEAE-cellulose chromatography. The MoAb was conjugated with the chelating agent DTPA using a modification of the method of Krejcarek and Tucker (11). Immediately prior to use, 1 mg of DTPA-coupled MoAb was mixed with 2.5 to 5 mCi '"In in aqueous HCI solution, and the reaction was terminated by an appropriate neutralizing buffer. Additional "cold" 96.5 was then added to achieve the desired MoAb concentration. The details of this method have been described in a previous paper (7). The immunoreactivity of 96.5 was assessed by Hybritech, Inc.,4 and ranged from 35 to 50%; it did not change following chelation as determined by the ability of 125l-labeled 96.5 to bind to excess solubilized antigen. Subjects. Thirty-one patients with biopsy-proven malignant melanoma received "'In-labeled 96.5 on the basis of an approved protocol. Sixteen males and 15 females with a median age of 40 years were studied. All patients gave written informed consent to the protocol in accordance with guidelines established by the Human Research Committee at M. D. Anderson Hospital and Tumor Institute. Patients were given a complete physical examination and had chest X-ray, electrocardiogram, complete blood counts, platelet counts, SGOT, lactic dehydrogenase, BUN, creatinine, and urinalysis performed. In addition, all patients had the appro4 Hybritech, Inc., unpublished data.

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MoAb IN MELANOMA priate radionuclide scans and/or computerized axial tomography scans necessary to document mÃ©tastases. All patients had measurable dis ease. The first 26 patients studied were skin tested with 0.1 ml (0.1 ng) of 96.5. Antibody was administered i.d. on the volar surface of the forearm along with a 0.9% NaCI solution (saline) control, and the area was examined at 6 and 24 h for evidence of a delayed-type hypersensitivity reaction. A positive skin test was defined as an area of induration at least 5 mm in diameter at 24 h. One patient who had received an Â¡mmunorestorative drug previously had a significant skin test reaction to 96.5 and for this reason was not given 96.5. All other patients had negative skin tests. Because of the lack of response to skin testing and the unpredictability of this procedure as confirmed by other investigators using 96.5,5 this test was dropped as a criterion to receive 96.5. Twentyone patients had sera collected and frozen at -20Â°C for subsequent determination of circulating antigen levels. Seven patients with accessible tumor had biopsies performed for immunohistological evaluation of p97 antigen expression. Study Methods. The first 23 patients entered on study received a single injection of 96.5 at concentrations of either 0.5 mg (one patient), 1 mg (4 patients), 2 mg (5 patients), 5 mg (5 patients), 10 mg (5 patients), and 20 mg (3 patients). During the dose escalation, the average radio activity of isotope was kept constant at 2.5 mCi. An additional 8 patients received 20 mg of MoAb and 5 mCi of1 "In in view of preliminary trials performed at Hybritech, Inc. (8), suggesting improved imaging at a higher mCi amount of 111ln.Vital signs were monitored at 15-min intervals during and for 2 h after MoAb administration. Complete blood counts, platelet count, SGOT, lactic dehydrogenase, BUN, creatinine, and urinalysis were repeated on Days 1 and 7 following infusion. Total-body imaging was performed using a longitudinal, tomographic imager (1 Phocon 192; Seiman Nuclear Imaging, Des Raines, IL). In some cases, patients were also imaged on a conventional ->camera with computer-assisted data storage (Gamma II; Digital, Maynard, MA). Back ground subtraction techniques were not used. Scans were read by a single nuclear medicine physician without former knowledge of disease sites. Measurement of Circulating Melanoma Antigen (p97). Antigen quantitation in patient serum was performed by Hybritech, Inc., using a previously published technique (4). Immunohistology. In selected cases where fresh tissue was available for evaluation, tumor expression of the p97 antigen was examined in vitro utilizing either standard immunoperoxidase or immunofluorescence techniques (19). Specimens were graded in intensity of peroxidase stain or fluorescence intensity from 1 to 4+ as compared to slides incubated with fluorescein or peroxidase-labeled irrelevant murine IgG, which served as a negative control.

RESULTS Tumor Imaging. The clinicalcharacteristics along with specific organ sites imaged for the 31 patients are listed in Table 1. Over one-half (58%) of the patients had received previous treatment with radiation or chemotherapy. Three patients had palpable lymph node recurrences within the regional drainage of the original primary (Stage III), whereas the remaining patients had one or more metastatic sites distant from the primary (Stage IV). There was a general trend toward an increase in the number of positive images by site as the total antibody concentration in creased. This was examined in greater detail by comparing the total number of mÃ©tastaseswhich imaged to the total number of detectable mÃ©tastasespresent at each dose level (Table 2). Of a total of 100 mÃ©tastaseswhich had been diagnosed previously by conventional techniques, 50 were visualized by 11lln scanning 5 D. J. Carlo, personal communication.

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for an overall true positive imaging rate of 50%. One patient (No. 31) had 2 areas of increased activity (upper spine and left scapula) which did not correspond with known mÃ©tastases.It is possible that these represented true areas of disease, although they also could have represented false positives. No other in stance of 111lnuptake in undocumented sites was found. Only 2 of 23 (9%) mÃ©tastaseswere visualized in patients receiving 2 mg of MoAb per 2.5 mCi of 111ln,whereas 25 of 31 mÃ©tastases (81%) were visualized in patients receiving 20 mg of MoAb per 5 mCi (P < 0.0001 ; x2 analysis). Optimal visualization of mÃ©tas tases appeared to occur above 5 mg of 96.5, with the exception that only 31% of metatases were imaged in 3 patients studied at the 20-mg/2.5-mCi dose (specific activity, 0.13 mCi/mg of 111ln).The number of mÃ©tastasesimaged increased with resto ration of specific activity to 0.25 mCi/mg of 111ln(20 mg/5 mCi). In the 16 patients given 10 and 20 mg of MoAb, the most frequent mÃ©tastasesimaged were skin (19 of 19 = 100%) and lymph nodes (8 of 9 = 89%). MÃ©tastasesless likely to take up tracer were bone (3 of 4 = 75%), lung (8 of 12 = 67%), and brain (4 of 12 = 33%). Liver mÃ©tastasescould not be discrimi nated from uptake of isotope in this organ. In several patients, however, space-filling defects in the liver were observed following 96.5 administration which persisted up until 144 h. There were no significant differences in tumor uptake of isotope in patients treated previously with chemotherapy or radiotherapy versus untreated patients. A greater percentage of mÃ©tastases larger than 1 cm in diameter imaged, although this was not significant at the P < 0.05 level (Chart 1). Large (>5 to 6 cm) necrotic mÃ©tastases,as determined on computerized axial tomography scan, did not image. Whole-Body and Region-of-lnterest Scans Obtained with 111ln.Representative images of several patients are shown in Figs. 1 and 2. Patient 26 had 2 recurrent s.c. nodules, one in the right breast and another over the left shoulder area. Immediately following infusion with 20 mg of MoAb labeled with 5.0 mCi of 111ln,a large amount of radioactivity was seen in the liver, spleen, and blood pool. By 48 h, blood pool radioactivity appeared to have decreased; however, a considerable amount of tracer was still present in liver and spleen with less 111lnseen in the heart and circulation. Uptake of 111lnin several tumor nodules could be seen (Fig. 1A). This radioactivity persisted in the tumor at 144 h (Fig. 1B) following a considerable washout of isotope from other organ sites except liver. Patient 29 (Fig. 2) had recurrent melanoma in the brain. Follow ing a 20-mg dose of MoAb, a 72-h scan demonstrated increased uptake of isotope within 2 parenchyma! mÃ©tastases. ToxicJty. Two patients who received 20-mg MoAb doses (Nos. 27 and 31) developed hives approximately 1 h after the infusion was begun. Both had relief of symptoms and gradual disappear ance of the rash following single i.v. doses of Benadryl and hydrocortisone; however, Patient 27 experienced a reexacerbation of symptoms necessitating discontinuation of infusion after receiving a total of only 15 mg of antibody. There were no episodes of wheezing, hypotension, or anaphylaxis observed. Moreover, there were no significant serial changes in hemoglobin or hematocrit, WBC count, platelet count, BUN, creatinine, SGOT, or urinalysis. Circulating Tumor Antigen. The level of free circulatingtumor antigen was measured in 21 patients from whom serum samples VOL. 45 MAY 1985

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MoAb IN MELANOMA Table 1 Clinical Parametersand Imaging Results

dose sites9_Â±â€”â€”+, Patient12345678910111213141516171819202122232425262728293031Age (yr)58313440313125765436264243293428534127647244612741376166592434SexMMMFMMFMMMFFMFFMFMMMFFMF treatmentsYesYesNOYesYesNoNoNoYesYesNoNoYesNONoYesYesYesYesYesYesYesYesNoYesNoNoYesYesNoNoDis sitesLiverLungBone, (mg/mCi)0.5/21.0/2.41.0/2.41.0/2.41.0/2.42.0/2.42.0/2.32.0/2.32.0/2

brainSkin, lung, nodesMediastini liver, brain, lung, lymph boneAxillarynodes, nodeLymph lymph nodesSkinLung,

onlyâ€”+â€”+, nodes

spineLymph lungSkinLungLymph nodes,

sites+, both sitesÂ±++, both

orbitLymph nodes, adrenals, brainSkinLymph nodes, skin,

sitesâ€”â€”++, all

nodeLymph nodeLung, abdomenLung, skin, brain, duodenum, adre nodesBrain, nals, lymph lungLymph brainLungBone, nodes, lung,

sites+, all nodes++, lymph

nodeLungLiverBreast, liver, lymph

only+â€”+, node

sitesâ€”+, all sites+, all

skinLymph sites++, both node(s)Lymph sites+, all skinLung, nodes, brainLymph skin, skinÂ±+, brain nodeBreast, lymph nodes, lung, brainMoAb/'"ln all sites * -, no uptake; +, uptake; Â±,equivocal uptake. Sites listed are only those where '"In uptake seen.

Table 2 Number of mÃ©tastases imaged in relation to dose of MoAb Dose of MoAb (mg)0.5

dose of '"In(mCi)2.0

1.0 2.0 5.0 10.0 20.0 20.0Mean

2.3 2.3 22 2.3 2.5 5.0Mean

specific activity of '"In (mCi/mg)4.0

of patients1

2.31.20.44

unknown mÃ©tastases"0

2(9) 4 23 0 10 5 5(50) 0 10 4(40) 5 0 12 9(75) 5 0 13 4(31) 3 0 31Imaged0(0)c 25 (81)Previously2 8MÃ©tastases*Known1

0.23 0.13 0.25No.

Total 31 100 50 (50) 8 In 3 patients, 2 at 1 mg, one at 2 mg, mÃ©tastases were too small and too numerous to count accurately. These were recorded by site rather than number. 6 Indicates uptake of tracer in areas (excluding liver, spleen, and bone marrow) not identified previously as mÃ©tastases. c Numbers in parentheses, percentage of imaged mÃ©tastases.

p97antigenwas demonstratedby immunohistologyin allcases, althoughconsiderablevariationin the intensityof tumorstaining wasobservedbetweenpatients.Cases11and30 demonstrated weak tumor stainingwith MoAb 96.5, and both patientshad equivocalin vivo tumor radioimmunodetection studies.Lymph nodemÃ©tastases wereimagedinPatients5 and7, andmoderate tumor-stainingintensityof lymphnodebiopsieswas observedin bothcases.Inthe 3 remainingpatients(Nos.4, 6, and8),tumor siteswere not imagedby radiolabeledMoAbdespitecleardem onstrationof tumor p97 antigenexpressionby Â¡mmunohistolog icalevaluations.An axillarylymphnode metastasisin Patient6 showedmoderatetumor-stainingintensitywith MoAb96.5 but was not imagedby radiolabeledMoAbin vivo.Similarly,skinand

couldbe obtained.Only2 of the 21 patients(Nos.3 and4) had serum antigenlevels barely above the upper limit of 2 ng/ml measuredin normal serum (2.4 and 4.4 ng/ml; respectively). Neitherof these patientsimagedfollowing1 mg of MoAb.The remainingpatientsall had levelsbetween0.5 and 2 ng/ml, the rangefoundin normalserum(datanot shown). Immunohistology. In 7 patients,freshtumortissuewasob tained for Â¡mmunohistological evaluationof tumor p97 antigen expression.Cryostat tumor sectionswere stainedwith MoAb 96.5, using standardindirectimmunoperoxidaseor immunofluorescencestainingmethods.Table3 summarizesthe immunohistologicalstainingresultsand the in vivo tumor radioimmunodetectionfindingsfor these patients.Tumor expressionof the CANCER

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MoAb

IN MELANOMA

10 . .

Tumor DlMMttr

(a)

â€¢sn:: â€¢ â€¢ Fig. 2. Patient with 2 metastatic lesions to the brain. Note uptake of "'In-labeled 96.5 (arrows). Scan performed at 72 h after administration of 20 mg MoAb per 5 mCi '"In.

ooo

o

Table 3 Tumor immunohistology and imaging results

Patient4 iMgcd

Chart 1. Comparison

lot

of size of metastasis

dose (mg/ mCi)1/2.4

biopsySkin of

p97 immunohistoof tu mor biopsy8Predose logyc4+ results8+

Inigtd

to uptake of '"In-labeled

96.5.

Individual mÃ©tastaseswhich did not image (O) were smaller in diameter than those which imaged (â€¢), although this was not statistically significant (P = 0.15; Student's f test).

other known sites of mÃ©tastaseswere not imaged in Patients 4 and 8, whose skin lesions displayed intense and moderate tumor staining, respectively. Interestingly, Patient 4 had an elevated serum p97 antigen level, and all 3 of these patients had received low doses (1 to 2 mg) of radiolabeled MoAb.

Predose 1/2.4 Lymph node 2+ 5 2/2.4 Lymph node Predose 2+ 6 Predose Skin 2+ 2/2.3 +Â± 7 Predose Skin 2+ 2/2.3 8 Predose Skin 1+ 5/2.2 11 1+ 20/4.8Site Lymph nodeTime PostdoseImagina Â±Tumor 30MoAb/'"ln " Utilized for immunohistology: Predose, prior to '"ln-96.5 infusion; Postdose, after '"ln-96.5 infusion. -, no uptake; +. definite uptake; Â±,equivocal uptake. c Intensity of tumor staining with MoAb 96.5 relative to background control irrelevant mouse IgG: 1, weak; 2, moderate; 3, strong; 4, very strong.

By increasing the total antibody concentration, large nontarget sites might be rapidly saturated by cold antibody, allowing for slower uptake of1 "In-labeled MoAb by tumor. Data which are

DISCUSSION The most significant findings in this study were that tumor imaging was readily achieved in 50% of patients, and more importantly, that imaging increased significantly as the dose of antibody increased, despite an overall decrease in specific activ ity of 111ln. The reason(s) for better imaging results obtained with larger doses of MoAb are obscure, although several hypotheses are tenable. Since the liver appears to avidly extract the MoAb and/ or 111lnfrom the circulation, the amount of circulating radiolabeled MoAb available to bind to tumor sites may be directly dependent on the amount given. The antigen, p97, is also expressed in low concentration on normal tissues compared to a relatively higher concentration on tumor (3). However, because of their compar ative size, normal tissues, particularly liver, may still contain more antigen sites which cross-react with 96.5, such as transferrin (2), or nonantigen sites, such as Fc receptors (20), available for rapid binding of 96.5 relative to the total sites available on the tumor.

supportive of this hypothesis are shown in Table 3. Three of 7 patients who did not image in vivo, yet had adjacent tumor tissue positive for p97 in vitro, had received less than 5 mg of antibody. These findings might argue that low tumor uptake of isotope was related to inaccessibility of enough circulating antibody, rather than the lack of antigen expression by the tumor. Currently, it is not known whether 20 mg of MoAb and 5 mCi of isotope are the highest concentrations needed for optimal imaging. A paradoxical decrease in number of sites imaged occurred at 20 mg/2.5 mCi. Although it is possible that the overall specific activity of 111lnwas too low to visualize MoAb uptake in tumor, it is also possible that imaging was related to other variables, such as tumor size or site of disease. For example, significantly more skin mÃ©tastases(see Table 1) were present in the 20-mg/5-mCi group. Hence, it may be premature to attribute changes in percentage of sites imaged solely to differences in amount of antibody administered. It is also pre-

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MoAb IN MELANOMA

mature to make comparisons using data from only 3 patients. More patients need to be studied to resolve these questions. Larson ef al. (12) found comparable imaging using 1-mg doses of a 131l-labeled anti-p97 MoAb which recognizes the epitope 8.2. However, it is difficult to make direct comparisons between the higher doses of MoAb used in this study and those used in Larson's study, since the characteristics of both MoAb and isotope were different. Furthermore, background subtraction techniques were not used in our study. Hence, these data suggest that the optimal imaging dose may need to be defined for each MoAb and isotope used. One patient (No. 31 ) had 2 areas (left scapula and spine) which imaged, yet which were not identified previously as metastatic sites of disease by other measures including bone scans. Unfor tunately, since these areas could not be confirmed by biopsy or other techniques, it is currently not possible to determine whether they represented previously undiagnosed metastasis or nonspe cific uptake of isotope. The uptake of 111lnin tumor and other tissues compares with previous animal studies which examined the relative merits of imaging with 111ln-labeled anti-CEA MoAb versus 125I.In the nude mouse model, 111ln-labeled anti-CEA MoAb increased gradually in tumor sites from 48 to 72 h after injection, whereas 125l-labeled anti-CEA had lesser accumulation in tumor and greater urinary excretion of isotope (21). Similar results have been demonstrated in a guinea pig model (1). As shown in Fig. 1, initial visualization of tumor occurred at 48 h. Tumor could still be faintly visualized as late as 144 h. Blood pool radioactivity appeared to be less at 144 than at 48 h, yet considerable isotope remained in the liver. In this study, there was a general trend for tumors less than 1 cm in size not to image, a finding which has been noted by Larson ef a/. (13) with 131l-labeled anti-p97 Fab fragments. Vari ability of antigen expression by tumor or accessibility of tumor antigen to antibody could also be involved as reasons for im proved imaging in soft tissue sites compared to lung, bone, or brain. The variation in intensity of tumor staining with 96.5 in vitro could also be reflective of variations in surface expression as a result of tumor heterogeneity (Table 3). A major limitation to the use of111 In-labeled 96.5 for in vivo imaging was the high background activity in liver, spleen, and bone marrow. Larson ef a/, using 131l-labeled96.5 Fab fragments observed more rapid clearing of isotope from these sites with 20 of 33 patients demonstrating positive scans, although quantita tive uptake of label by tumor was less (13). Several additional radioimaging trials in humans using MoAb have been reported. Mach ef al. (14), using 131l-labeled MoAb to colon carcinoma, observed a 51% positive imaging rate with intact antibody and a slightly higher percentage (61%) using F(ab')2 fragments. In a study of "'In-labeled anti-M8 (breast carcinoma) MoAb, Rainsbury ef al. (17) found variable uptake of isotope in bone compared to ^c-diphosphonate. Uptake was not observed in other tumor sites; however, the actual dose of MoAb administered was less than 5 mg in all cases. Future studies using radiolabeled MoAb for tumor imaging need to be directed toward elucidating the mechanisms responsible for nontumor uptake as well as improv ing imaging techniques. ACKNOWLEDGMENTS The authors wish to acknowledge Christopher Hofeditz, Dianna Martin, James Myers, Robin Starr, Linda Smith, Joanne Fabiano, Mammen John, and Nancy Moldawer, for expert technical assistance and Ruth Wang for manuscript prepa ration.

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REFERENCES 1. Bernhard, M. I., Hwang, K. M., Foon, J. M., Keenan, A. M., Kessler, R. M., Frincke, J. M., Tallam, D. J., Hanna, M. G., Jr., Peters, L., and Oldham, R. K. Localization of 111ln- and 125l-labeled monoclonal antibody in guinea pigs bearing line 10 hepatocarcinoma tumors. Cancer Res., 43: 4429-4433,1983. 2. Brown, J. P., Hewick, R. M., HellstrÃ¶m, l., HellstrÃ¶m, K. Eâ€žDoolittle, R. F., and Dreyer, W. J. Human melanoma-associated antigen P97 is structurally and functionally related to transferrin. Nature (Lond.), 296:171-173,1982. 3. Brown, J. P., Woodbury, R. G., Hart, C. E., HellstrÃ¶m,I., and HellstrÃ¶m, K. E. Quantitative analysis of melanoma-associated antigen P97 in normal and neoplastic tissues. Proc. Nati. Acad. Sci. USA, 78: 539-543,1981. 4. David, G. S., Present, W. A., Martinis, J., Wand, R., and Bartholomew, R. M. Monoclonal antibodies in the detection of hepatitis infection. J. Med. Lab. Sci., 38:341-348,1981. 5. Goldenberg, D. M., Kim, E. E., and Deland, F. 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The clinical evaluation of "'In-labeled monoclonal anti-melanoma antibodies ("'In Anti-mel) for human scanning. J. NucÃ-.Med., 24: 15,1983. 9. HellstrÃ¶m, I., Brown, J. P., and HellstrÃ¶m,K. E. Monoclonal antibodies to two determinants of melanoma-antigen P97 act synergistically in complement dependent cytotoxicity. J. Immunol., 727: 157-160, 1981. 10. Kim, E. E., Deland, F. H., Nelson, M. O., Bennett, S., Simmons, G., Alpert, E., and Goldenberg, D. M. Radioimmunodetection of cancer with radio-labeled antibodies to a-fetoprotein. Cancer Res., 40: 3008-3012,1980. 11. Krejcarek, G. E., and Tucker, K. L. Covalent attachment of cheating groups to macromolecules. Biochem. Biophys. Res. Commun., 77: 581-585,1977. 12. Larson, S. M., Brown, J. P., Wright, P. W., Carrasquillo, J. A., HellstrÃ¶m, I., and HellstrÃ¶m, K. E. Imaging of melanoma within 1-131-labeled monoclonal antibodies. J. NucÃ-.Med., 24: 123-129,1983. 13. Larson, S. M., Carrasquillo, J. A., Krohn, K. A., Brown, J. P., McGuffin, R. W., Ferens, J. M., Graham, M. M., Hill, L. D., Beaumier, P. L., HellstrÃ¶m, K. E., and HellstrÃ¶m, I. Localization of '3'l-labeled P97-specific Fab fragments in human melanoma as a basis for radiotherapy. J. Clin. Invest., 72:2101-2114, 1983. 14. Mach, J. P., Chatal, J. F., Lumbroso, J. D., Buchegger, F., Forni, M., Ritschard, J., Berche, C., Douillard, J. Y., Carrel, S., Heriyn, M., Steplewski, Z., and Koprowski, H. Tumor localization in patients by radiolabeled monoclonal anti bodies against colon carcinoma. Cancer Res., 43: 5593-5600,1983. 15. Pressman, D., Day, E. D., and Blau, M. The use of paired labeling in the determination of tumor localizing antibodies. Cancer Res., 17: 845-850,1957. 16. Pressman, D., and Keighley, G. The zone of activity of antibodies as determined by the use of radioactive tracers: the zone of activity of nephritoxic anti-kidney serum. J. Immunol., 59:141-146,1948. 17. Rainsbury, R. M., Westwood, J. H., Coombes, R. C., Neville, A. M., Ott, R. J., Kalirai, T. S., McCready, V. R., and Gaset, J. C. Location of metastatic breast carcinoma by a monoclonal antibody chelate labeled with indium-111. Lancet, 2:934-938, 1983. 18. Rosenblum, M. R., Murray, J. L., Haynie, T. P., Glenn, H. J., Jahns, M. F., Benjamin, R. S., Frincke, J. M., Carlo, D. J., and Hersh, E. M. Pharmacokinetics of "'In-labeled anti-P97 monoclonal antibody in patients with metastatic malig nant melanoma. Cancer Res., 45: 2382-2386,1985. 19. Sobol, R. E., Dillman, R. O., Smith, J. D., Imai, K., Ferrane, S., Shawler, D., Glassy, M. C., and Royston, I. Phase I evaluation of murine monoclonal antimelanoma antibody in man: preliminary observations. Hybridomas in cancer diagnosis and treatment. Progr. Cancer Res. Ther., 21:199-206,1982. 20. Steplewski, Z., LÃ¼beck,M. D., and Koprowski, H. Human macrophages armed with murine immunoglobulin G2. antibodies to tumor destroy human cancer cells. Science (Wash. DC), 227: 865-867,1983. 21. Stern, P., Hagan, P., Halpem, S., Chen, A., David, G., Adams, T., Desmond, W., BrÃ¤utigam, K., and Royston, I. The effect of the radiolabel on the kinetics of monoclonal anti-CEA in a nude mouse-human colon tumor model. Hybri domas in cancer diagnosis and treatment. Progr. Cancer Res. Ther., 27:199206, 1982. 22. Woodbury, R. G., Brown, J. P., Loop, S. M., HellstrÃ¶m, K. E., and HellstrÃ¶m, I. Analysis of normal neoplastic human tissues for the tumor-associated protein P97. Intern. J. Cancer, 27. 145-149,1981. 23. Woodbury, R. G., Brown, J. P., Yeh, M. Y., HellstrÃ¶m, I., and HellstrÃ¶m, K. E. Identification of a cell surface protein, P97, in human melanomas and certain other neoplasms. Proc. Nati. Acad. Sci. USA, 77: 2183-2186,1980 24. Yeh, M. Y., Hellstrom, I., Abe, K., Hakomori, S., and HellstrÃ¶m, K. E. Cell surface antigens of human melanoma identified by a monoclonal antibody. Proc. Nati. Acad. Sci. USA, 76: 2927-2931, 1979.

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1A

1B Fig. 1. A, anterior (left) and posterior (right) total-body ->-scanperformed 48 h after MoAb infusion (20 mg MoAb per 5 mCi isotope). Note considerabledistribution of isotope in blood pool, heart, gastrointestinal tract, liver, spleen,bone marrow, and nasopharynx.Arrows, uptake in metastatic s.c. nodules in right breast and left shoulder posteriorly. B, repeat scan at 144 h; considerable radioactivity still remains in liver. Arrows as in A. CANCER

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