[CANCER RESEARCH 56, 2123-2129. May 1, 1996]

Comparative Metabolism and Retention of Iodine-125, Yttrium-90, and Indium-IllRadioimmunoconjugates by Cancer Cells1

Oliver W. Press,2 Darning Shan, Janet Howell-Clark, Janet Eary, Frederick R. Appelbaum, Dana Matthews,David J. King, Adrian M. R. Haines, Philip Hamann, Lois Hinman,3 Daniel Shochat,4 and Irwin D. Bernstein

Departments of Medicine (Division of Oncology} ¡O.W. P., D. S., J. H-C., F. R. AJ. Pediatrics [D. M., I. D. B.¡.Radiology ¡J.E.I, and Biological Structure ¡O.W. P., D. S.,J. H-C.I of the University of Washington, the Fred Hutchinson Cancer Research Center [O. W. P., F. R. A.. D. M., 1. D. B.], Seattle, Washington 98195-6043: Celltech, Ltd.,Slough United Kingdon [D. J. K.. A. M. R. H.¡:and Wyeth-Ayerst Research. Pearl River, New York 10965 [P. H., L H., D. S.]

ABSTRACT

Radiolabeled antibodies have produced encouraging remissions in patients with chemotherapy-resistant hematological malignancies; however,

the selection of therapeutic radionuclides for clinical trials remains controversial. In this study, we compared the internalization, lysosomal targeting, metabolism, and cellular retention of radiolabeled murine andhumanized monoclonal antibodies targeting the CD33 antigen (monoclonal antibodies ml'67 and hP67, respectively) on myeloid leukemia cell

lines (HEL and III,-60) and of anti-carcinoma antibodies (monoclonal

antibodies hCTMOl and hA33) targeting breast cancer and colorectalcarcinoma cell lines (MCF7 and Colo 205, respectively). Each antibodywas labeled with I25I (by the lodoGen method) and with '"In and ""V

using macrocyclic chelation technology. Targeted tumor cells were analyzed for retention and metabolism of radioimmunoconjugates using cellular radioimmunoassays, Percoli gradient fractionation of cell organelles,SDS-PAGE, and TLC of cell lysates and culture supernatants. Our results

suggest that antibodies are routed to lysosomes after endocytosis, wherethey are proteolytically degraded. [125I]monoiodotyrosine is rapidly ex

creted from cells after lysosomal catabolism of antibodies radioiodinatedby conventional methods, whereas small molecular weight "'In and '"'Y

catabolites remain trapped in lysosomes. As a consequence of the differential disposition of small molecular weight catabolites, '"In and '"'V

conjugates displayed superior retention of radioactivity compared withI25Iconjugates when tumor cells were targeted using rapidly internalizing

antibody-antigen systems (e.g., hP67 with HEL cells and hCTMOl withMCF7 cells). When tumor cells were targeted using antibody-antigensystems exhibiting slow rates of endocytosis (e.g., hP67 on 111,-60 cells and

hA33 on Colo 205 cells), little difference in cellular retention of radioactivity was observed, regardless of whether I2SI, lnln, or *°Ywas used.

INTRODUCTION

Systemic chemotherapy has achieved limited success in eradicatingdisseminated malignancies, emphasizing the need for innovative newapproaches to cancer therapy. Recent clinical trials with radiolabeledMoAbs5 directed against tumor-associated antigens have demon

strated considerable promise (1-6); however, many uncertainties re

main concerning the optimal implemention of this new modality. Oneof the most contentious issues remains the selection of the optimalradionuclide for clinical trials. Iodine-131 has been the radiolabel used

in the majority of studies to date because of its ready availability, lowcost, simple radiochemistry, and long track record of success in curingthyroid carcinoma. In addition, the highest reported response rates,complete response rates, and longest response durations reported with

Received 11/3/95; accepted 2/27/96.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 in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

'This work was supported by DOE Grant DE-FG06-92ER6I459. N1H POI Grant

CA44991. and a grant from the American Cyanamid Corporation.2 To whom requests for reprints should be addressed, at University of Washington

Medical Center, P.O. Box 356043, Seattle. WA 98195-6043.1 Present address: Immunomedics. Inc., Morris Plains, NY.4 Present address: Coulter Pharmaceutical. Inc., 550 California Avenue, Suite 200,

Palo Alto. CA 94306-1440.5 The abbreviations used are: MoAb, monoclonal antibody; ATCC, American Type

Culture Collection; TCA. trichloroacetic acid.

radioimmunoconjugates have been observed in clinical trials using131I as the therapeutic nuclide (1, 2, 4).

On the other hand, several limitations of 13II have been defined. Its

energetic y emissions pose a risk to health care personnel and contribute to myelosuppression. Furthermore, I-labeled antibodies pre

pared by conventional chloramine T or lodoGen methods are rapidlydegraded after endocytosis by target cells, with rapid release of131I-labeled tyrosine from cells after lysosomal proteolysis (7-13).

Several investigators have suggested that alternative radionuclidesmay prove superior to I31I for radioimmunotherapic applications,

including alternative ßemitters (e.g., yttrium-90, rhenium-186. andcopper-67) and a-emitters (e.g., astatine-211 or bismuth-212). Although little consensus has emerged regarding the optimal radioiso-tope, 90Y has been proposed most often as a replacement for I3II (14,15). Favorable characteristics of 90Y include its lack of y emissions,

high energy ßemissions, and superior tumor retention (14-17). It iswidely assumed that the favorable retention of 90Y in tumor cells isdue to prolonged retention of 90Y inside target cell lysosomes, in amanner analagous to that reported for "'In ( 11. 13. 18, 19). However,

few studies supporting this contention have been published (13). Thispoint merits direct experimental confirmation, since differences in thebehavior of indium and yttrium in vivo have been reported (20-22),

and because it has been demonstrated that some radiometals, such asrhenium-188, are rapidly excreted from tumor cells after lysosomal

targeting (18).In this report, we compare and contrast the internalization, metab

olism, and retention of 125I, "'In, and 90Y-labeled antibodies target

ing malignant human tumor cell lines. Our experiments initiallyfocused on the behavior of radioimmunoconjugates prepared with theanti-CD33 MoAb P67 because of its potential usefulness in treating

patients with myeloid leukemias (23). Previous studies demonstratedrapid internalization and metabolism of 13'I-labeled murine P67 by

the HEL cell line in vitro, by HEL xenografts in nude mice, and bymyeloid leukemia cells in patients in vivo (23, 24). Rapid expulsion ofradioiodine from leukemic cells after endocytosis of 131I-labeled

mP67 resulted in poor retention of radioactivity and the consequentdiscontinuation of clinical trials using 131I-labeled mP67 prepared bythe chloramine T method (23). Novel 131I-labeled mP67 constructs

prepared using the nonmetabolizable tyramine cellobiose carbohydrate adduci improved cellular retention of I, but the limitedspecific activity achievable with this approach has hindered therapeutic applications (23, 24).

In the present report, cellular radioimmunoassays and Percoli gradient fractionation of cell organelles were used to test whether theretention of radioactivity targeted to tumor cells using MoAbs couldbe improved by using radionuclides such as '"in and 90Y in place of131I,and to determine whether the improved retention of radioactivity

was due to lysosomal trapping of the radiometals. SDS-PAGE andTLC were used to test whether retained cell-associated radioactivity

was present on intact radioimmunoconjugates or on catabolized fragments. Our experiments showed that retention of radioactivity bytumor cells was better for ' "in and 90Y than 131Iif rapidly internal-

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MLTABOI.ISM 01 RADIOIMMI NOCONJUGATES

¡zingantibodies were used but that little difference was observed ifantibodies undergoing minimal endocytosis were used. Cell fraction-

ation studies confirmed trapping of small molecular weight catabolitesin lysosomes and suggested that the cellular processing of '"in andy"Y were similar.

MATERIALS AND METHODS

Cell Suspensions. The human myeloid leukemia cell lines. HEL (from Dr.Paul Martin. Fred Hutchinson Cancer Research Center, Seattle. WA) andHL-60 (from the ATCC). and the Colo 205 colon carcinoma cell line (fromATCC) were maintained in log-phase growth in RPMI 1640 (Life Technolo

gies, Inc., Grand Island. NY) supplemented with 12% PCS, 2 mM glutamine,and 1 niM pyruvate at 37°Cin 5% carbon dioxide. The MCF7 breast carcinoma

cell line (from ATCC) was grown in DMEM (BioWhittaker, Walkersville,MD) with 10% PCS, 100 units/ml penicillin. 100 fig/ml streptomycin, 2 mMglutamine. and 5 ng/liter insulin. The viability of cells ranged from 90-98%

by trypan blue exclusion in the experiments described. A series of controlexperiments demonstrated that trypsinization of the adherent Colo 205 andMCF7 cell lines prior to plating in microtiter plates did not diminish thebinding of radiolabeled HA33 or hCTMOI antibodies, respectively.

Antibodies. Antibodies used in these experiments included humanizedanti-CD33 MoAb hP67 (IgG4), humanized anti-mucin antibody hCTMOI(IgG4), and humanized anti-carcinoma MoAb hA33 (IgGl), which were

prepared and purified by Celltech, Ltd. (Berkshire. United Kingdom) bymethods published previously (25-29). Protein concentrations were deter

mined using the BCA assay following the recommendations of the manufacturer (Pierce Chemical Co., Rockford. IL).

Radioiodination. MoAbs were iodinated by incubating 100 /j.g MoAb with0.5 mCi of Na['25I] (Amersham) in glass tubes coated with 10 fig of lodoGen(Pierce Chemical Co.) for 10 min at room temperature. Free [I25I] was

removed by chromatography on a Pharmacia PD-10 column (Pharmacia,

Piscataway, NJ).Radiometal Conjugation. Antibodies were conjugated to the proprietary

homobifunctional macrocyclic cross-linking reagents. 9N3-maleimide (for'"In) and 12N4-maleimide (tetra-azocyclododecane tetra-acetic acid, for '"Y)

via thioether bonds under metal-free conditions as described previously (28.29). ""'Y labeling was carried out on preparations dialysed with 0. l M potas

sium acetate buffer (pH 6) at antibody concentrations >\ mg/ml (29). [9"Y]C1,

(0.42 mCi; Amersham) was added to 0.14 mg antibody in 200 ¡JL\0.1 Mpotassium acetate buffer (pH 6), and the preparation was incubated at roomtemperature for 15 min. The labeling was then quenched by the addition of 20/xl of 50 mM diethylenetriaminepentaacetic acid (final concentration, 5 mM),followed by further incubation at room temperature for 10 min. The labelingefficiency was assessed by TLC and TCA precipitation, and generally approximated 90%. Free ""'Y was removed by desalting on a PD-10 column (Phar

macia); <\% of radioactivity added to cells was in the form of free metal.

Antibody immunoreactivities were measured by the method of Lindmo et al.(30) and were found to be 95-100% for 125I -hP67 and 69-96% for ""Y-

labeled hP67 in the preparations used for these experiments. Antibody aviditieswere measured by the method of Badger et al. (31) and found to be 5.7 X IO9liters/M for I25l-labeled hP67 and 2.7 X 10" liters/M for '"Y-labeled hP67.

Cell Binding and Retention of Antibodies. Target cells were washed incold, serum-free tissue culture medium and pelleted by centrifugation. MoAbsthat had been trace-labeled with '-'I, '"In, or""Y were added to the cell pelletsin a ratio of 150 ng protein per H)6 cells in a volume of 0.5 ml. Cells wereincubated with radiolabeled MoAbs at 4°Cfor l h and then washed twice withtissue culture medium at 4°C. Nonspecific binding was assessed by pre-

incubating control cells with a 100-fold excess of nonradioactive antibodybefore incubating with radiolabeled antibodies. Aliquots containing 1 x IO6

labeled cells were plated in 200 /j.1 RPMI 1640 in microtiter plates (FlowLaboratories, McLean, VA), warmed to 37°Cin a humidified 5% CO2 incu

bator for 0, 1,4, 18, and 24 h, and then assayed for cell-associated radioactivity

and supernatant radioactivity by gamma counting (Beckman Gamma 7000Counter; Palo Alto, CA). Nonspecific binding of labeled MoAbs as tested byblocking with "cold" (nonradioactive) antibody was generally <5% of total

bound radioactivity, (although in 1 of 18 experiments, it was 16% for 1-125-hP67 on HL-60 cells). Both Bremstrahlung emissions and ßemissions were

quantified for W)Yusing gamma counting and scintillation counting, respectively. Curves obtained by the two counting methods for '"'Y-labeled MoAbs

were superimposable.Antibody Degradation. The extent of degradation of the radiolabeled

MoAbs was measured as described (32, 33). Culture supernatants (0.2 ml)were mixed with 0.6 ml of 25% TCA to precipitate protein-bound radioactivity

released from the cell surface. Precipitates were washed with 0.5 ml 25% TCA,and the radioactivity in the pellets (TCA-insoluble) and supernatants (TCA-

soluble) was determined as above.Percoli Gradient Fractionation of Organdíes. Cells were incubated at

4°Cwith saturating concentrations of radiolabeled MoAbs, washed twice, andthen incubated at 37°Cin a humidified incubator (5% CO2) for 0-24 h. Cellaliquots (50 x IO6 cells) were then suspended in TES buffer (10 mM trietha-

nolamine, 0.25 M sucrose, pH 7.5). disrupted with 25 strokes of a Douncehomogenizer (Kontes. Inc., Molina, IL), and sedimented for 10 min at 250 X gto remove nuclei and unbroken cells. The supernatatant (1 ml) was layered onthe surface of a 20% solution of Percoli in TES buffer (9 ml) and centrifugedat 4°Cfor 60 min at 20,000 x g. Serial 0.5-ml fractions were collected and

assayed for radioactivity and for lysosomal /3-galactosidase activity as

described previously (33).SDS-Polyacrylamide Electrophoresis. The metabolism of radiolabeled

MoAbs was assessed by solubilizing cells in 0.1% NP40 after 0, 4, or 24 h ofincubation at 37°C,mixing with protease inhibitors (leupeptin, 0.05 mg/ml;

aprotonin, 0.02 mg/ml; pepstatin, 0.07 mg/ml; and phenylmethylsulfonyl fluoride, 4 mM), centrifuging at 10,000 X g to remove insoluble material, mixing1:1 with sample buffer, boiling, loading onto 10% polyacrylamide gels containing 0.08% bisacrylamide, and performing electrophoresis as described byLaemmli (34). Processed radioactive gels were exposed to Kodak X-Omatic

film in holders equipped with DuPont Cronex intensifying screens to produceautoradiographs.

TLC. The radioactive molecular species present in the acetone-soluble

portion of cell culture supernatants were characterized by TLC by the methodof Geissler et al. (1. 8).

Statistics. Cellular radioimmunoassays were performed with triplicate replicates, and the means and SEs were calculated. Differences were comparedusing the two-tailed, unpaired f test.

RESULTS

Metabolism of 125I-, "'In-, and "°Y-labeledhP67 (anti-CD33)

Antibodies by HEL Cells. The degradation and retention of theanti-CD33 MoAb hP67 was studied after conjugation to 125I, '"in,and 90Y using the human erythroleukemia cell line, HEL. 125I-labeled

hP67 was rapidly internalized by HEL cells and degraded with therelease of small molecular weight, TCA-soluble catabolites to theculture supernatant (Fig. la). By 24 h of incubation at 37°C,53 ±5%

of the initially bound radioactivity had been processed by cells andreleased to the culture medium in TCA-soluble form. TLC demonstrated that >75% of TCA-soluble radioactivity derived from 125I-

labeled MoAbs in such cultures was present in the form of mono-

iodotyrosine (data not shown). A small fraction (11 ±3%) of intiallybound radioactivity was released from cells in intact, TCA-precipita-ble form. SDS-PAGE electrophoresis demonstrated that the TCA-

precipitable supernatant radioactivity was almost entirely composedof intact hP67 immunoglobulin (data not shown). As might be expected, total cell-bound radioactivity declined progressively during

culture, with only 35 ±1% of initially bound radioactivity remainingcell associated after 24 h of culture. Ten concordant experimentsdemonstrated the reproducibility of these observations.

The disposition of '"In-labeled hP67 (Fig. \b) and 90Y-labeled

hP67 (Fig. le) after binding to HEL cells differed in several respectsfrom that of 125I-labeled hP67. Most notably, 66 ± 1% of '"in-labeled hP67 and 70 ±2% of 90Y-labeled hP67 remained cell associated after 24 h of culture, compared to only 35 ±1% of 125I-labeledhP67 (P < 0.00001 for 125I versus either '"in or 90Y; Fig. 2). The

superior cellular retention of the radiometal conjugates was primarily

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METABOLISM OF RADIOIMMUNOCONJUGATES

^^^~* Cdl assoc.cpm"""O Supt. cpm- -*r •¿�TCA sol. cpm-•-*••-TCApplcpm

8 12 16 20 24100,

80

60

40

20

In-lll-P67

„¿�•¿�.4-:«

:-::::-;

o 12 16 20 24

100

60

40

20-

Y-90-P67

„¿�..........-•*o"" '."

ofr*--y0 8 12 16 20 24

HOURSFig. 1. Internalizaron and degradation of I25l-labeled (a), '"In-labeled (b), and

•¿�"Y-labeled(c) anti-CD33 MoAb hP67 by the HEL leukemia cell line. HEL cells (IO6)

were incubated with 150 ng of trace-radiolabeled hP67 at 4°C,washed, placed in freshculture medium, and incubated at 37°Cfor 0, 1,4, 18, and 24 h before quantifying the

amounts of cell-associated (•)and supernatant (O) radioactivity by gamma counting.Supernatant radioactivity was fractionated into small molecular weight, TCA-soluble (A)and larger, TCA-precipitable (A) components. SE bars are plotted for triplicate replicates

but are too small to be visualized for most time points.

a result of diminished exocytosis of small molecular weight, TCA-soluble catabolites from cells binding '"in- and 90Y-labeled hP67

(9 ±1% and 5 ±1% of culture radioactivity, respectively, after 24 hof culture versus 53 ±5% with 125I-labeled hP67; P < 0.0002 for 125Iversus either '"In or ^Y; Fig. 2). Interestingly, hP67 labeled with'"in or 90Y was more prone to passive dissociation or "shedding"from HEL cells than 125I-labeled hP67, with TCA-precipitable super

natant radioactivity accounting for 25 ±2% of total culture radioactivity with the radiometals, compared with 11 ±3% for 125I-labeledhP67 (P < 0.015 for 125I versus either '"In or 90Y). Enhanced

shedding of the metal-chelated antibodies compared with the radio-

iodinated antibody was consistently observed in six consecutive

experiments and has been reported by other investigators (18). Asnoted by Shih et al. (18), the shedding of intact radiolabeled MoAboccurred early during the culture period, reaching maximal levels by4 h of incubation and then plateauing (Fig. 1).

Comparative Metabolism of I2SI-, '"In-, and ""Y-labeled hP67

Conjugates by a Cell Line with a Slower Rate of Endocytosis(HL-60). Our experimental results with the rapidly internalizinghP67-HEL system suggested that radiometal conjugates might pro

vide superior retention of targeted radiotherapy than that observedwith 131I-labeled hP67. To assess whether this observation pertained

to cell lines with slower rates of endocytosis, we compared theinternalization and degradation of the three radioimmunoconjugatesby the HL-60 leukemia cell line, which was known to internalizeanti-CD33 antibodies more slowly than HEL cells. Although thediminished release of small molecular weight, TCA-soluble catabolites was again observed with '"in- and '"Y-labeled hP67 comparedwith 125I-labeled hP67, the magnitude of the differences was small

and only reached statistical significance after 18-24 h of culture (Fig.3). Because of greater shedding of intact TCA-precipitable ' ' 'in- and90Y-labeled hP67 compared with 125I-labeled hP67 (data not shown),

retention of cell-associated radioactivity after 4 h was actually superior for cells labeled with 125I-labeled hP67 (84 ± 1%) than with

0 4 8 12 16

HOURSFig. 2. Cell-associated and TCA-soluble supernatant radioactivity in cultures of HEL

cells incubated for 0, 1,4, 18, or 24 h after surface labeling with 125I-labeled hP67 (•),'"In-labeled hP67 (A), or ""Y-labeled hP67 (O) as described in the legend to Fig. 1.

12 16HOURS

Fig. 3. Cell-associated and TCA-soluble supernatant radioactivity in cultures of HL-60cells incubated for 0, 1,4, 18, or 24 h after surface labeling with ':5I-labeled hP67 (•),'"In-labeled hP67 (A), or *°Y-labeledhP67 (O) as described in the legend to Fig. 1.

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METABOLISM OF RADIOIMMUNOCONJUGATES

30-1

UJ 20 H

u. 10-o

HELOh

30 i

20-

10-

HEL

1-125

0369 12151821 0369 12151821

iJ

fhO

20-1

10-

HL-60

20-1

lO-i

HL-60

0 3 6 9 12151821

Fraction

0 3 6 9 12151821

FractionFig. 4. Transit of '"In-labeled and '25I -labeled anti-CD33 MoAb HP67 from Ihe cell

surface (fraction 2) to lysosomes (fractions 17-19) in HEL cells (a and c) and HL-60 cells(b and d) as demonstrated using Percoli gradient fractionation of cell organdíes. Cellswere surface labeled with I2*I- or '"In-labeled hP67, washed and incubated for 0 h (O),

8 h (•).or 24 h (D) before disrupting cells in a Dounce homogenizer and separating cellorgandíes by buoyant density using continuous Percoli gradients. The location of lysosomes was confirmed using the enzyme marker, 0-galactosidase (not shown).

'"In-labeled hP67 (78 ±1%) or ^Y-labeled hP67 (71 ±2%) after4 h of incubation (P < 0.001 for I25I versus ulln or ""Y; Fig. 3).

However, because shedding of intact hP67 conjugates occurredmainly in the first 4 h of culture, whereas metabolism and exocytosisof small molecular weight TCA-soluble 125I-labeled catabolites began

at 4 h and progressively increased, the cell retention patterns weresimilar for all three radioimmunoconjugates by 24 h of culture(Fig. 3). The overall areas under the cell-retention curves were notsignificantly different for any of the three conjugates using HL-60cells as the target cell line (Fig. 3). Seven experiments with HL-60

cells demonstrated similar findings.Intracellular Location of Radioimmunoconjugates. To deter

mine the intracellular location of internalized radioimmunoconjugates, HEL or HL-60 cells were surface-labeled with 125I-, ' ' 'in-, or'"Y-labeled hP67 antibodies at 4°C,washed, incubated for varioustime periods at 37°C,and disrupted with a Dounce homogenizer; then

cell organelles were separated by Percoli gradient centrifugation.These experiments documented the progressive transfer of radioimmunoconjugates from low density surface membrane fractions to highdensity lysosomal fractions (Fig. 4), as confirmed by colocalization of/3-galactosidase activity (data not shown; Refs. 7, 8, and 48). Becauselow molecular weight I25l-labeled catabolites were rapidly released

from lysosomes to the culture medium whereas low molecular weight'"in- and ^Y-labeled catabolites were retained inside lysosomes

(Refs. 11, 13, and 18; Fig. 1), the lysosomal accumulation of radio-

metals was consistently more striking than the cumulative accretion of125I in this organelle (Fig. 4, compare a and b with c and d).

Comparative Degradation and Retention of "'In- and I2SI-

labeled MoAbs by Carcinoma Cells. To assess the relevance of ourobservations for the radioimmunotherapy of solid tumors, we analyzed the comparative retention of '"in- and I25l-labeled antibodies

targeting the MCF-7 breast cancer cell line (MoAb hCTMOl) and the

Colo 205 colorectal carcinoma cell line (MoAb hA33). The hCTMOlantibody was readily internalized and degraded by MCF7 cells, resulting in the release of more radioactivity to the supernatant ofcultures of 125I-labeled hCTMOl cells (58 ±4% after 24 h) than incultures of '"in-labeled hCTMOl cells (34 ±3%; P < 0.02). As aconsequence, cellular retention of '"in-labeled hCTMOl was significantly better than retention of the 125I-labeled conjugate (66 ±1%

versus 42 ±2% after 24 h; P < 0.001 Fig. 5a).In contrast, the hA33 conjugates were slowly internalized by the

Colo 205 cell line, resulting in minimal release of radioactivity to thesupernatant with either '"in- or 125I-labeled conjugates. Accordingly,cellular retention of both reagents was excellent (82 ±1% for '"In-labeled hA33 and 89 ±2% for 125I-labeled hA33 after 24 h; Fig. 5b).

Analysis of intracellular trafficking by the Percoli gradient methodconfirmed lysosomal targeting of hCTMOl with progressive accumulation of '"in in lysosomes (Fig. 6). As in the myeloid leukemiamodel, lysosomal accretion of 125I was less impressive than that of

100i .

1-125 Sup.In-Ill Sup.

1-125 Crii Assoc.

In-Ill Oil Auor

8 12 16 20 24

60

40-

hA33on Colo205

12 16HOURS

Fig. 5. a, quantification of cell-associated and supernatant radioactivity in cultures ofthe MCF-7 breast cancer cell line after surface-labeling with either 125l-labeled or'"In-labeled MoAb hCTMOl and incubating for 0, 1,4, 18, or 24 h at 37°C.(•,I25Icell-associated radioactivity; •¿�125I supernatant radioactivity; O, '"In cell-associatedradioactivity; D, '"In supernatant radioactivity). SE bars are plotted for triplicate replicates but are too small to be visualized for most time points. /'•.quantification of

cell-associated and supernatant radioactivity in cultures of the Colo 205 colon carcinomacell line after surface-labeling with either '"[-labeled or '"In-labeled MoAb hA33 andincubating for 0, 1,4, 18, or 24 h at 37°C.(•,I25I cell-associated radioactivity; •¿�I25Isupernatant radioactivity; O, '"In cell-associated radioactivity; D, '"in supernatant

radioactivity). SE bars are plotted for triplicate replicates but are too small to be visualizedfor most time points.

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METABOLISM OF RADIOIMMUNOCONJUOATES

MCF-7

0369 12151821

FRACTION

0 3 6 9 1215182 1

FRACTIONFig. 6. Transit of ' "in-labeled and 125I-labeled MoAbs hCTMOl (a and c) and hA33

(b and d) from the cell surface (fraction 2) to lysosomes (fractions 17-19) in MCF7 cells

(a and c) and Colo 205 cells (b and d) as demonstrated using Percoli gradient fractionationof cell organelles. Cells were surface-labeled with 125I-or '"in-labeled MoAbs, washed,

and incubated for 0 (O), 8 (•),or 24 h (D) before disrupting cells in a Douncehomogenizer and separating cell organelles by buoyant density using continuous Percoligradients. The location of lysosomes was confirmed using the enzyme marker, ß-galac-tosidase (not shown).

"'In, due to the rapid excretion of TCA-soluble, small molecularweight 125I-labeled metabolites (Fig. 6). Percoli gradient experiments

confirmed the minimal endocytosis of hA33 conjugates by Colo 205cells under the conditions used, as indicated by the negligible accumulation of radioactivity in high-density lysosomal fractions. In con

trast to our findings with hA33 on Colo 205 cells, Welt et al. (35) havereported that murine A33 is readily endocytosed by some other coloncancer cell lines, e.g., SW1222.

DISCUSSION

Six conclusions can be derived from the experiments reported inthis study: (a) cellular retention of 1HIn and '"Y was superior to

retention of radioiodine when radionuclides were targeted to tumorcells with antibodies undergoing rapid endocytosis (>20% of antibody internalized within 4 h); (b) retention of "'In, 90Y, and radio-

iodine were similar when targeted to tumor cells using MoAbs, whichwere slowly internalized; (c) 125I-labeled MoAbs were rapidly de

graded in lysosomes to monoiodotyrosine (7, 8) and other low molecular weight catabolites, which were rapidly expelled from cells; (d)radiometals were retained intracellularly, even after trafficking tolysosomes. Consequently, little TCA-soluble radioactivity accumu

lated in the supernatants of tumor cells targeted with radiometalconjugates; (e) radiometal conjugates were "shed" in intact form to agreater degree than comparable 125I-labeled conjugates; and (f) "'In-and 90Y-labeled antibodies appeared to be internalized, shed, metab

olized, and excreted identically by tumor cells, using the methods ofanalysis used in this study.

These findings confirm and extend reports from other investigatorsdescribing superior intracellular retention of '"In-labeled antibodiescompared with 123I-labeled MoAbs (13, 18, 36, 37). More impor

tantly, this report is one of the first to directly demonstrate the cellularprocessing and disposition of 90Y-labeled MoAbs by tumor cells (13).This demonstration is important, because 90Y has been promulgated

as the best radionuclide for therapeutic clinical trials (14, 15) andbecause previous work has demonstrated variable intracellular processing of different radiometal nuclides, with protracted lysosomalretention reported for '"in (11, 13, 18) and 67Cu (38) but rapidexocytosis for l88Re (18). Our findings suggest that the cellularprocessing of "'in and 90Y by tumor cells is similar, validatingextrapolations of 90Y behavior based on prior observations with '"In

conjugates (11, 13, 18). Our experiments also confirm the enhancedrate of "shedding" of radiometal conjugates compared with radioio-

dinated conjugates reported by Shih et al., (18), possibly due to a2-fold difference in measured antibody avidity between 125I-labeledhP67 and 90Y-labeled hP67 (5.7 X IO9 liters/M and 2.7 X IO9 liters/M,

respectively). Shih et al. (18) have hypothesized that antibodies possessing critical lysine residues in the antigen-combining site may havediminished antibody avidity after radiometal chelation using lysine-

directed methods as opposed to tyrosine iodination, leading to a highershedding rate. In spite of the higher shedding rate, overall cellularretention of rapidly internalized antibodies was superior after labelingwith '"in or 90Y compared with 125Idue to markedly greater intra

cellular retention of the former.Although we believe that our conclusions are well supported by our

data and by the literature, caution must be exercised to avoid over-

interpretation. These experiments have addressed only the retention ofradionuclides by tumor cells in vitro and do not test their comparativeretention by normal tissues (e.g. liver, kidney, and bone) in vivo.Animal studies indicate that the retention of '"in and 90Y is pro

longed in normal organs such as bone, liver and kidney as well as intumor sites (24, 39-41), partially mitigating the salutary effects of

enhanced tumor retention of radiometal conjugates. Nevertheless,several model systems have suggested enhanced tumonnormal organratios of retained radioactivity using '"In and 90Y, especially when

internalizing antibodies are studied (14, 24, 42, 43).Our studies do not assess the impact of the different ßparticle

energies and path lengths of 90Y and 131Ion radioimmunotherapeutic

efficacy. It is theoretically conceivable that the higher maximal ßparticle energy (2.2 versus 0.6 MeV) and longer maximal path lengthof 90Y (5.3 versus 0.8 mm) may yield superior radioimmunotherapeutic effects, even if the retention of 90Y and 131I-labeled MoAbs areidentical. Conversely, the enhanced bone and liver retention of 90Y-

labeled conjugates may result in increased myelosuppression andhepatotoxicity compared with 131I-labeled conjugates.

It is important to recognize that several strategies besides the use ofradiometal nuclides may be used to augment cellular retention ofradioimmunoconjugates. Deliberate selection of noninternalizing antibodies targeting surface-stable antigens has been shown to success

fully circumvent lysosomal degradation of MoAbs and to producesuperior cellular retention of 131I-labeled conjugates (24, 44). Thisapproach has been employed using 13ll-labeled anti-CD20 antibodiesfor B-cell lymphomas and 131I-labeled anti-CD45 antibodies for my-

eloid leukemias with excellent clinical results (1-4). A second alternative is to employ novel radioiodination techniques using nonme-tabolizable carbohydrate adducts [i.e., "residualizing" moieties such

as tyramine cellobiose (45, 46), N-succinimidyl 3-(tri-n-butylstannyl)benzoate (47), or dilactitol-'25I-labeled tyramine (12, 18)]. These131I-labeled conjugation methods produce 131I-labeled carbohydrate

intermediates, which are retained in lysosomes after internalization ina manner similar to the radiometals because the radioiodinated carbohydrate moieties cannot be fully degraded and exocytosed (12,45-47). Clinical adoption of this approach for therapeutic applica

tions has, unfortunately, been retarded by limitations in the specific

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METABOLISM OF RADIOIMMUNOCONJUGATES

activity that can be obtained with these novel residualizingml-labeled carbohydrate conjugates using current methodologies.

A third alternative involves the systemic administration of lysoso-

motropic amines (e.g., chloroquine and amantadine), carboxylicionophores (monensin and nigericin), or calcium channel blockers(verapamil), which have been shown to inhibit lysosomal degradation and exocytosis of 125I-labeled MoAbs (48). In vitro thesemethods permit a doubling of the retention half-time of 125I-

labeled MoAbs by tumor cells (48); however, currently availabledrugs have proven to be too toxic at the requisite concentrations forsafe clinical application.6

Rigorous evaluation of the relative clinical merits of conventionalI31l-labeled MoAb conjugates, novel residualizing 131I-labeled con

jugates, radiometal conjugates, and comparisons of internalizing andnoninternalizing antibodies will require comparative biodistributionstudies in cancer patients. Only a single study has been reportedaddressing these issues in a controlled scientific fashion. In this report,patients with cutaneous T-cell lymphomas received infusions of arapidly internalizing 131I- or " 'In-labeled anti-CD5 MoAb T101,

followed by serial quantitative gamma camera imaging (43). Onlyfour patients were studied with 13ll-labeled T101, and only tworeceived infusions of "'In-labeled T101, but both achieved betterbiodistributions with '"in-labeled T101 than with 131I-labeled T101.

In our opinion, more studies with larger patient numbers will berequired to assess the relative merits of the various radiolabelingstrategies and the relative attractiveness of internalizing and noninternalizing antibodies. Because large inter-patient variations in tumorburden, tumor MoAb uptake and retention, serum half-lives, and

MoAb catabolic rates have been repeatedly observed (49), we believethat the best strategy for such studies in lymphoma patients will be thesequential administration of trace-labeled MoAb conjugates prepared

by various methods on successive weeks, followed by serial quantitative gamma camera imaging, tumor and normal tissue biopsies, andestimation of absorbed radiation doses to tumor sites and normalorgans. We have demonstrated previously the feasibility of administering up to five sequential trace-labeled infusions in patients with

lymphomas ( 1) and believe that such studies will be crucial in settlingcurrent controversies concerning the optimal radionuclide-antibody

combinations for clinical cancer therapy. Sequential antibody administration studies will be more difficult to perform in patients with solidtumors, due to the rapid formation of human antimouse antibodies thatalter antibody pharmacokinetics and biodistributions.

ACKNOWLEDGMENTS

We gratefully acknowledge the secretarial assistance of Patricia Kury Adamand the technical assistance of Carol Dean and Caroline Thostenson.

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1996;56:2123-2129. Cancer Res   Oliver W. Press, Daming Shan, Janet Howell-Clark, et al.   CellsYttrium-90, and Indium-111 Radioimmunoconjugates by Cancer Comparative Metabolism and Retention of Iodine-125,

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