effectoftumormassandantigenicnatureonthebiodistributionofl...

(CANCER RESEARCH 49, 2884-2889, June 1, 1989]

Effect of Tumor Mass and Antigenic Nature on the Biodistribution of LabeledMonoclonal Antibodies in MiceYuji Watanabe, Keigo Endo,1 Mitsuru Koizumi, Yasutaka Kawamura, Tsuneo Saga, Harumi Sakahara,

Masahide Kuroki, Yuji Matsuoka, and Junji KonishiDepartment of Nuclear Medicine. Kyoto University Hospital, Shogoin, Sakyo-Ku, Kyoto 606 fY. W., K. E., M. K., Y. K., T. S., H. S., J. KJ, and Department ofBiochemistry, School of Medicine, Fukuoka University, Fukuoka 812 [M. K., Y. M.], Japan

ABSTRACT

The effect of tumor mass and antigenic nature on the biodistributionof '"In- and 125I-labeled monoclonal antibodies (MoAbs) was studiedusing F(ab')i fragments of three representative anti-tumor MoAbs and

SVV1116 human colorectal carcinoma grown in nude mice. The 19-9,F33-104 anti-CEA, and 17-1A MoAbs showed specific binding toSW1116 cells. The former two MoAbs recognize circulating CA 19-9with molecular weights of more than 5,000,000 and CEA of M, 170,000-180,000, respectively, whereas 17-1A reacts with a nonshedding antigen.Both percentage injected dose per gram tumor and tumor-to-blood ratioswere inversely proportional to the tumor mass in nude mice administered'"In- and I25l-labeled 19-9, but liver uptake increased as tumor size

increased. Analysis of serum samples and tumor homogenates demonstrated the presence of a high-molecular-weight species, probably due tothe antibody binding to CA 19-9. In the case of '"In-labeled anti-CEA

MoAb, tumor uptake also decreased and liver uptake increased withtumor size, but this effect was less obvious than that of 19-9. In contrast,tumor and liver uptake of I25l-labeled anti-CEA MoAb, '"In- and I25I-labeled 17-1A and control antibodies were independent of tumor mass.The absolute tumor uptake and tumor-to-blood ratios of all I25l-labeledantibodies were lower than those of the '"In-labeled ones. And the effectof tumor mass was also weaker with I2sl-labeled antibodies, probably due

to in vivo dehalogenation. These results indicate that the effect of tumorsize on the incorporation of labeled MoAb into tumors is dependent onthe antigenic nature to be targeted and/or radionuclides used for labelingand that high concentrations of circulating high molecular weight antigensmay limit in vivo use of MoAb conjugates.

INTRODUCTION

The development of hybridoma technique has made the production of MoAbs2 feasible, and MoAbs have been employed

for a variety of purposes, including the diagnosis and therapyof malignancies in humans (1-8). Radionuclides and chemo-therapeutic agents are conjugated with MoAbs to target cyto-toxic agents to tumor cells for direct killing. Such an approachhas the advantage of increasing tumorcidal activity and decreasing the systematic toxicity of these cytotoxic agents. Theseapplications require the knowledge of pharmacokinetics andbiodistribution of labeled MoAbs after administration to patients, since both the sensitivity of immunoimaging and theeffect of therapy using MoAb conjugates are dependent on theabsolute tumor uptake and/or on the tumor-to-normal tissueratios of labeled MoAbs (9-18). The outcome of therapy willbe estimated from the absolute tumor uptake, the tumor weightand the response to cytotoxic agents. However, the biodistribution of MoAb conjugates are greatly influenced by manyfactors, such as the nature of antigens to be targeted, antibodyor its fragments, radionuclides, coupling methods, the size oftumor and so on (10-18).

Received 8/8/88; revised 1/18/89; accepted 2/21/89.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.

1To whom requests for reprints should be addressed, at Department of NuclearMedicine. Kyoto University Hospital, Shogoin, Sakyo-ku, Kyoto 606, Japan.

2The abbreviations used are: CEA, carcinoembryonic antigen; MoAb, mono

clonal antibody; DTPA, diethylenetriaminepentaacetic acid.

There have been conflicting findings concerning the relationship between the tumor size and the uptake of antibody conjugates administered i.v. (12-15). In the interest of establishingthe effect of tumor mass, the nature of antigens and radionuclides on the biodistribution of MoAb conjugates, we haveextended this investigation to include well-characterized threeMoAbs reactive with gastrointestinal tumors, 1116-NS-19-9,anti-CEA, and CO 17-1A. These MoAbs recognize differentrepresentative cancer-associated antigens, CA 19-9, CEA, and17-1A, respectively, all of which were expressed on humancolorectal cancer cells SW1116 (19-28). F(ab')z fragments labeled with '"In- and radioiodine were administered to nude

mice bearing SW1116 tumors by the same route and at thesame dose, to avoid nonspecific binding of Fc portion of MoAbsto Fc receptors and to increase tumor-to-normal-tissue ratiosby the rapid clearance from circulation.

MATERIALS AND METHODSMonoclonal Antibodies. Rah')., fragments of murine MoAb 1116-

NS-19-9 (IgG,), CO 17-1A (IgG2a), and anti-CEA (F33-104) (IgG,), aswell as normal murine immunoglobulin controls were used in thepresent study (19-26). Both uncoupled and DTPA-coupled F(ab')2fragments of MoAb 1116-NS-19-9 and CO 17-1A were provided fromCentocor (Malvern, PA) through the courtesy of Nihon MediphysicsCo. (Takarazuka, Japan). These three MoAbs are known to be reactivewith SW1116 human colorectal carcinoma cells (19, 25-28). F(ab')2

fragments of normal murine immunogloblin were supplied by NihonMediphysics. MoAb 1116-NS-19-9 recognizes a sialylated fucopen-taose II carbohydrate determinant, designated CA 19-9 expressed oncirculating glycoprotein antigens with molecular weight of more than5,000,000 (21, 22, 28) and anti-CEA MoAb (F33-104) is reactive withthe CEA-specific protein part of CEA with molecular weight of170,000-180,000 (23, 24). CA 19-9 and CEA are representative cancer-associated antigens released into circulation, whereas 17-1A is reportedto react with a nonshedding antigen (25, 26).

Radiolabeling. Antibodies were readily labeled with '"Invia chelationwith DTPA by simply incubating DTPA-coupled F(ab')2 fragments and["'Injchloride for 30 min, at room temperature, and the labeling

efficiency was more than 90% without any further purification steps.The chloramine T method was employed for the radio-iodination ofMoAb, as described previously (29). The specific activity of radio-labeled MoAb was between 0.5 and 2 mCi/mg for '"In-labeled antibodies, between 4 and 7 mCi/mg for I25l-labeled antibodies and between1.5 and 3 mCi/mg for '-"I-labeled antibodies.

The immunoreactive fractions of radio-labeled F(ab')2 fragments of

MoAb 19-9, 17-1A, and F33-104 were calculated as about 0.60, 0.60,and 0.40, respectively, according to the method of I.indino et al. (30).The affinity constants for 19-9, 17-1A, and F33-104 to SW1116 cellswere determined as 1.5 x 10", 0.5 x 10", and 1.3 x 10"/M, respectively,

by the Scatchard plot analysis (29).Tumors. BALB/c nude mice were xenografted with human colorectal

cancer cell line SW1116 by implanting 5 x IO6 cells s.c. in their left

flank (28). Tumors were allowed to grow for 2 to 12 weeks until theyreached the desired size. SW1116 tumors usually grew to l g in about5 to 7 weeks after injection and had only very small necrotic zones.Thyroids were blocked with drinking water containing 0.1% w/v iodo-potassium from one day before to the end of the study. The serum of

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EFFECT OF TUMOR MASS ON BIODISTRIBUTION OF LABELED MONOCLONAL ANTIBODIES

xenografted nude mice was obtained by retro-orbital puncture two daysbefore the injection of radio-labeled MoAbs. Serum CA 19-9 and CEAconcentrations of the mice were determined at dilutions of 1:2 and 1:5for CA 19-9 and without dilution for CEA, using commercially availablekits (CIS, Saclay, France and Daiichi Radioisotope Laboratories, Tokyo, Japan) (24, 31).

Biodistribution and Imaging. Nude mice bearing SW1116 xenograftswere given i.v. injection of'"In-(l pd) and 125I-(1^Ci) labeled F(ab')2

fragments. The antibody dose was adjusted to 20 ^g per mouse bymixing labeled and unlabeled MoAb. Tumors weighing 0.1-2.5 g wereused in the present study. Animals were sacrificed 48 h after theadministration and the tissues were weighed and counted. The biodis-tribution data were represented as a percentage of the injected dose pergram normalized to a 20-g mouse and tissue-to-blood ratios.

Statistical analysis was performed using linear regression analysisand variance analysis.

Animal images were made using a gamma-camera (Searle, Chicago,IL), equipped with a pinhole collimator at 6, 24, and 48 h followingthe administration of 100 ÃŸdof I3'l-labeled MoAbs.

Chromatographie Analysis. Various samples were analyzed by gel-filtration chromatography in the search for chemical forms of I25Iin

the samples. The injectate, serum samples of nude mice and solublefractions of liver and tumor homogenates of nude mice (supernatantsobtained by centrifugation at 100,000 x g for 60 min) were applied toa Sephacryl S-300 column (1.6 x 95 cm) equilibrated in 50 mM PBS,pH 7.5. Fractions were collected and the eluted radioactivity wascounted with a well gamma-counter.

RESULTS

Tumor Mass and Serum Antigen Concentrations. In order toestablish the relationship between tumor size (weight) and

concomitant CA 19-9 and CEA release from SW1116 cells innude mice, serum samples were obtained from mice bearingSW1116 tumors, and serum CA 19-9 and CEA concentrationswere determined as described in "Materials and Methods." As

reported by Klug et al. (28), there was a significant correlationbetween the size of tumors and serum CA 19-9 values in nudemice. When serum CA 19-9 levels in units/ml was plottedagainst tumor mass in grams, the correlation coefficient was0.72, the slope was 59.97, and the y intercept was 16.6, usinglinear regression analysis (P < 0.001) (data not shown). Circulating CEA levels also correlated well with the tumor size (r =0.71, P < 0.001) (data not shown).

Effect of Tumor Mass on Tumor Uptake. '"In- and 125I-labeled F(ab')2 fragments of 19-9, anti-CEA, and 17-1A MoAb

showed specific localization in SW1116 tumors grown in nudemice. Mean tumor-to-blood ratios of these '"In-labeled MoAbs

were 33.4 (n = 25), 5.5 (n = 10), and 13.4 (n = 12), respectively,at 48 h after the administration. Tumor uptake expressed aspercentage injected dose per gram tumor and tumor-to-bloodratios of all '"In-labeled MoAbs were higher than those of

radioiodinated ones (Figs. 1 and 2). As shown in Fig. I/I, innude mice administered with '"In-labeled 19-9 and anti-CEA

MoAb, the tumor uptake was inversely proportional to thetumor size (r = 0.65, P < 0.01 and r = 0.59, P < 0.01,respectively). But the effect was more intense with 19-9 (slopeof-6.40) than with anti-CEA MoAb (slope of-0.98), and thetumor uptake of '"In-labeled 17-1A did not demonstrate a

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SW1116 tumor weight (g)Fig. 1. Relationship between SW1116 tumor size and tumor uptake of percentage injected dose per gram tumor of '"In- (A) and '"I- (B) labeled F(ab')2 fragments

of 19-9 (top left), anti-CEA (top right), 17-1A (bottom left) and control (bottom right) antibodies.

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SW1116 tumor weight (g)Fig. 2. Relationship between SW1116 tumor size and tumor-to-blood ratios of '"In- (A) and 125I-(B) labeled F(ab')2 fragments of 19-9 (top left), anti-CEA (top

right), 17-1A (bottom left) and control (bottom right) antibodies.

significant correlation with tumor size. As shown in Fig. 2A,tumor-to-blood ratios of "'In-labeled 19-9 and anti-CEA Mo Abalso decreased as tumors enlarged (r = 0.45, P < 0.05, slope of-10.93 and r = 0.42, P < 0.05, slope of -1.21, respectively)and tumor-to-blood ratios of "'In-labeled 17-1A and control

antibodies were independent of tumor size.The effect of tumor size on tumor uptake was also seen with

125I-labeled 19-9 (Figs. IB and 2B). Both the percentage injecteddose per gram and tumor-to-blood ratios of radio-iodinated 19-9 decreased as tumor size increased (r = 0.54, P < 0.01, slopeof -1.11 and r = 0.47, P < 0.05, slope of -8.21, respectively).But the effect was less than that of '"In-labeled 19-9 (slope ofâ€”6.40and â€”10.93,respectively). Tumor uptake and tumor-to-blood ratios of '"I-labeled anti-CEA, 17-1A and control anti

bodies were found to be independent of tumor size.Effect of Tumor Mass in Normal Organs. In normal organs

including the intestine, stomach, lung, muscle, and bone, withthe exception of kidney which excreted radiolabeled metabolicproducts, '"In- and '"I-labeled F(ab')2 fragments of 19-9, anti-CEA, and 17-1A Mo Ab showed neither specific localizationnor tumor size dependency (data not shown). However, '"In-and 125I-labeled F(ab')2 fragments of 19-9 were incorporated

into liver and spleen more than those of the other three antibodies. The accumulation of "'In-labeled 19-9 and anti-CEAMoAb in liver increased with tumor size (r = 0.81, P < 0.001,slope of 14.50 and r = 0.50, P< 0.05, slope of 1.60, respectively)(Fig. 3/0. Liver-to-blood ratios of '"In-labeled 19-9 also increased as tumors enlarged (r = 0.59, P < 0.01, slope of 48.09),

showing greater effect than that of anti-CEA MoAb (r = 0.55,P < 0.01, slope of 3.66) (Fig. 4A). This effect of tumor masson liver uptake was less with 125I-labeledMoAb than with "'In-

labeled MoAbs (Figs. 3 and 4). A similar effect of tumor masson spleen uptake was also seen (data not shown).

Scintigrams. Scintigrams of nude mice following i.v. injections of 131I-labeled F(ab')2 fragments of 19-9, F33-104, and17-1A MoAbs confirmed the results of the biodistributionstudies (data not shown). All three MoAbs visualized theSW1116 tumors xenografted in nude mice at 24 and 48 h afteradministration. High liver uptake was observed in nude miceadministered with 19-9, but not with either anti-CEA or 17-1AMoAbs.

Chromatographie Analysis. Various samples including 125I-labeled F(ab')2 fragments of 19-9, serum of nude mice, and liverand tumor homogenates were evaluated by Sephacryl S-300gel-chromatography (Fig. 5). A radiolabeled high molecularweight peak appeared in the void volume in serum samples,and liver and tumor homogenates. More high molecular weightform was demonstrated in tumor homogenates than in liverhomogenates, indicating the presence of an antigen-antibodycomplex. In addition, the majority of radioactivity in liverhomogenates was not the labeled antibody in the injected formbut a lower molecular weight species.

DISCUSSION

The principal object of this study was to examine the effectof tumor mass, radionuclide and antigenic nature on the tumor

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Fig. 3. Relationship between SW1116 tumor size and liver uptake of percentage injected dose per gram liver of '"In- (A) and '"I- (B) labeled F(ab')2 fragmentsof 19-9 (lop left), anti-CEA (top right), 17-1A (bottom Â¡eft)and control (bottom right) antibodies.

uptake, and biodistribution of labeled Mo Abs. SW1116 humancolorectal carcinoma cell line transplanted in nude mice wasselected as a tumor model, because it expressed three representative cancer-associated antigens, CA 19-9, CEA, and 17-1A (19, 25-28). Nonimmunological factors that would affectthe entry and handling of antibodies by tumor, such as vascu-

larity, necrosis, lymphatic drainage, and extracellular space(32), would apply equally to tumor-specific 19-9, anti-CEA,and 17-1A MoAbs and control antibody, and the selectiveuptake of the former three MoAbs by the tumor seemed to bemainly attributable to the antigen-binding properties of the

MoAbs.An inverse correlation between tumor mass and uptake in

the tumor, as well as a proportional increase in liver and spleenuptake with tumor size has been demonstrated in nude miceinjected with '"In- and '"I-labeled 19-9, and '"In-labeled anti-

CEA MoAbs, while these effects were not observed with the17-1A or control antibodies. The major difference between theformer two MoAbs (19-9 and anti-CEA) and the 17-1A MoAbwas that CA 19-9 and CEA are circulating antigens while 17-1A antigen is nonshedding. We have found a high molecularweight species in the serum and tumor homogenates, probablydue to antibody binding to CA 19-9 by gel-chromatographicanalysis (10, 11). Once circulating antigens form intravascularantigen-antibody complexes, then they are removed by thereticuloendothelial system, which may result in a decreasedtumor uptake and increased liver uptake of labeled antibodies

(11-15). The effect of tumor mass was also observed with '"In-labeled anti-CEA MoAb, but less obvious than with 19-9. CA19-9 is a mucin with molecular weight of more than 5,000,000(21, 22), while that of CEA is A/r 170,000-180,000 (23, 24).The antigenic nature to be targeted, such as a release into thecirculation system or the difference of molecular weight, maybe one of the reasons for different effects of tumor mass on thebiodistribution of labeled MoAbs. However, other factors, suchas crossreactivity of 17-1A with normal tissues (19), tissuepermeability and so on, could influence the biodistribution insome way.

Similar effect was observed with '"In-labeled anti-CEAMoAb, but not with I25l-labeled one. It may be presumably due

to rapid deiodination or the release of radioiodine from theMoAb, which would also explain why the tumor size effect of'"In-labeled 19-9 was stronger than with '"I-iabeled one (13,

33).In the present study, instead of intact MoAb, we employed

F(ab')2 fragments of four antibodies to avoid a nonspecificuptake caused by the Fc receptor, and a high tumor-to-normal-tissue ratio was reached by a rapid blood clearance of theradioactivity (17). Furthermore, we administered 20 pg of antibodies to each nude mouse bearing SW1116 tumor. In recentclinical trials, 20 to 100 mg of antibodies were used by addingun labeled MoAbs for the diagnosis and therapy of tumors, sincethe tumor detection rate increased as the antibody administeredincreased (1, 2, 8). The 20 Mg of antibody per mouse wasequivalent to this range.

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right), 17-1A (bottom left) and control (bottom right) antibodies.

SW1116 tumor weight (g)'In- (A) and 125I-(B) labeled F(ab')2 fragments of 19-9 (top left), anti-CEA (top

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(top right), soluble fractions of tumor (bottom left) and liver (bottom right)homogenates of SW1116 bearing nude mice at 48 h after injection, a, voidvolume; b, IgG; c, F(:ib'). fragment; d, albumin; e. Fab' or Fab fragment;/, low

molecular weight species larger than free iodine; , radioactivity; , opticaldensity at 280 nm.

Clinically, high liver deposit is often considered to be problematic in the use of "'In-labeled antibodies (11). However,

surprisingly, liver uptake of labeled MoAbs varied, althoughsome properties of the '"In radiolabcl itself were similar. Thehighest liver uptake was seen with 19-9, as previously reported(11) and low with anti-CEA and 17-1A MoAbs. Liver metastasis was detected in some patients with colon cancer usinglabeled anti-CEA and 17-1A MoAbs (7, 8), though it may bemasked by an intense liver uptake of labeled 19-9 Mo Ab.

Therapeutic applications of MoAb conjugated with radio-nuclides, toxins, or antineoplastic drugs will be also dependenton the results shown here. For example, the radiation doseestimate for tumor and normal tissues is directly proportionalto the percentage injected dose per gram of tissue (34, 35).Yttrium-90, which is labeled for MoAbs through the chelationwith DTPA, has been described as one of the best radionuclidesfor tumor therapy when conjugated with MoAbs (36, 37). Inthe case of 19-9 and DTPA-coupled anti-CEA MoAb, onewould anticipate that smaller tumor could be given more radiation doses per unit tumor size with less toxicity to liver. Furthermore, if serum human anti-mouse antibody titer of a patientis high due to repeated injections of MoAb conjugates, theadministered MoAb may be removed in the form of immunecomplexes from circulation, resulting in less tumor uptake (38).These results indicate that the effect of tumor size on theincorporation of labeled MoAb into tumors is dependent onthe antigenic nature to be targeted and/or radionuclides used

2888




for labeling and that high concentrations of circulating highmolecular weight antigens may limit in vivo use of MoAbconjugates.

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1989;49:2884-2889. Cancer Res Yuji Watanabe, Keigo Endo, Mitsuru Koizumi, et al. Biodistribution of Labeled Monoclonal Antibodies in MiceEffect of Tumor Mass and Antigenic Nature on the

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