[CANCER RESEARCH 48, 1823-1828, April 1, 1988]

Comparison of Tumor Targeting of Mouse Monoclonal and Goat PolyclonalAntibodies to Carcinoembryonic Antigen in the GW-39 HumanTumor-Hamster Host Model1

Robert M. Sharkey, F. James Primus, Dan Shochat, and David M. Goldenberg2

Center for Molecular Medicine and Immunology [R. M. S., F. J. P., D. M. G.J at the University of Medicine and Dentistry of New Jersey, and Immunomedics, Inc.ID. S.I Newark, New Jersey 07103

ABSTRACT

We have evaluated 4 radioiodinated mouse monoclonal anticarcinoem-bryonic antigen antibodies (MAbs) by using the GW-39 human colorectal

tumor xenograft transplanted i.m. in immunocompetent hamsters todetermine whether there were any differences in their tumor localizationproperties. Additional comparisons were made to affinity-purified goat

anticarcinoembryonic antigen antibody. Statistically significant differences were found in the percentage/g of tumor uptake and tumor/nontu-iiuir ratios among the antibodies, so that the antibodies could be rankedaccording to their tumor localization properties (NP-2 > NP-4 = goatantibody > NP-1 > NP-3). Although statistical differences were found,

tumor/nontumor values generally were not distinguished by a factor ofmore than 1.5, suggesting that these differences may not be biologicallysignificant. F(ab')2 fragments of NP-2 were found to be superior to NP-4 F(ah'), fragments, giving tumor/liver and tumor/blood ratios of 16 and

11.5, respectively, within 3 days, in comparison to 5.4 and 3.8 for NP-4F(ab'}z fragments. Mixtures of all of the MAbs or a mixture of NP-2

and NP-4 did not improve tumor localization, in comparison to NP-2

alone. These studies suggest that mixtures of these anticarcinoembryonicantigen MAbs may not afford better tumor imaging than the use of acertain single antitumor MAb.

INTRODUCTION

Radiolabeled antibodies directed against a diverse number ofhuman tumors have been used to locate tumors by externalscintigraphic methods (1-9). In the process of generatingMAbs,3 several hybridoma clones may be identified that rec

ognize distinctly different epitopes on the same antigen molecule. Hedin et al. (10) and Buchegger et al. (11) have reportedon the localization properties of several MAbs against CEAand have found that certain of these MAbs localized colonietumor xenografts in nude mice better than others. We havecharacterized 4 MAbs against CEA (12). NP-1 is a MAb thatrecognizes an epitope shared with NCA, an antigen also foundon human granulocytes, and another antigen identified in nic-conium, that may represent a precursor to CEA (12). NP-2 andNP-3 recognize distinct epitopes that are shared with CEA andmeconiuni antigen, but are not NCA. NP-4 recognizes anepitope that is only expressed on CEA. All of the NP-seriesMAbs are IgGi, but they differ in their affinity binding constantsin a range from 8.9 x 10" to 5.3 x 10" M~' (12).

Since these MAbs recognize distinct epitopes on CEA andby in vitro testing have different affinities for CEA, we wantedto compare the tumor targeting properties of these MAbs in anin vivo model. In this report, we have compared these MAbsagainst each other, as a mixture, and against an affinity-puri-

Received 3/30/87; revised 10/22/87; accepted 12/29/87.The costs of publication of this article were defrayed in pari 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.

1Supported in part by NIH Grants CA 37218 and OÕACA 39841.2To whom requests for reprints should be addressed, at Center for Molecular

Medicine and Immunology, 1 Bruce St., Newark, NJ 07103.3The abbreviations used are: MAb, mouse monoclonal antibody; CEA, carci-

noembryonic antigen; NCA, nonspecific cross-reacting antigen.

fied, polyclonal goat anti-CEA antibody, using a human colontumor xenograft-hamster host model. In addition, we havestudied the tumor localization properties of F(ab')2 fragments

prepared from 2 of the MAbs.

MATERIALS AND METHODS

Antibodies. The initial characterization of these MAbs has beendescribed previously (12). All of the MAbs were isolated from ascitesfluid by a single passage through a protein A immunoadsorbent. Polyclonal goat anti-CEA was prepared by affinity Chromatograph), asdescribed by Primus et al. (13). Two separate lots of affinity-purifiedgoat antibody were prepared. One lot was processed over an NCA anda CEA immunoadsorbent, while the other lot of goat antibody wasprocessed only over the CEA immunoadsorbent. F(ab')2 fragments

were prepared by pepsin digestion, followed by desalting on a columnof Fractogel (TSK-HW-50F; EM Science, Gibbstown, NJ) and by ionexchange chromatography (DE-52 cellulose, Whatman, or S-Sepha-rose, Pharmacia, Piscataway, NJ). AH antibody preparations (intactIgG and F(ab')i fragments) were tested for purity by immunoelectro-

phoresis, by sodium dodecyl sulfate-polyacrylamide gel electrophoresisof unreduced and /3-mercaptoethanol-reduced samples, and by sizeexclusion high pressure liquid chromatography on Micropak TSK 3000SW (7.5 x 300 mm; VarÃ-an,Walnut Creek, CA).

The antibodies were radioiodinated with I3II (New England Nuclear,

North Billerica, MA) by the chloramine-T method (14) to a specificactivity of 12-15 mCi/mg. An irrelevant mouse myeloma IgG, desig

nated Ag8 (P3x6 Ag8), was isolated from mouse ascites by protein Aand radioiodinated with I25Iin a similar fashion. Normal goat IgG was

purchased from PelFreeze (Rogers, AR), and was used without furtherpurification as an irrelevant IgG for the studies with goat anti-CEAantibody. Unbound radioiodine was separated from antibody-boundiodine by gel filtration over a PD-10 (Pharmacia) column equilibratedin 0.04 M phosphate-buffered saline, pH 7.4, containing 1% human

serum albumin. Before injection into animals, all radiolabeled antibodies were characterized by molecular sieve chromatography over an S-

200 (Pharmacia) column (1.6 x 60 cm), and for immunoreactivity bypassage through a CEA immunoadsorbent as well as an anti-mouseIgG or anti-goat IgG immunoadsorbent. In all instances there was lessthan 1% aggregated antibody and, except for NP-4, less than 1% smallmolecular weight radioiodine. Radiolabeled NP-4 preparations had 2-

4% small molecular weight radioiodine. The percentage of binding ofthe radiolabeled MAbs to a CEA immunoadsorbent was 75, 75, 95, 80,and 65 for NP-1, NP-2, NP-3, NP-4, and the goat anti-CEA antibody,

respectively. There was no difference found in the binding to the CEAimmunoadsorbent of the goat antibody prepared by the 2 methodsmentioned above. However, the one lot of goat antibody that was notpurified by passage over an NCA immunoadsorbent bound 35-40% to

an NCA immunoadsorbent, whereas the goat antibody that was purifiedby passage over both the NCA and CEA immunoadsorbents only boundbetween 8 and 12% to the NCA immunoadsorbent. The F(ab')2 frag

ments of NP-2 and NP-4 bound 90 and 80%, respectively, to a CEA

immunoadsorbent. Greater than 90% of all of the radiolabeled antibodies bound to their respective anti-mouse or anti-goat IgG immunoadsorbents. Radiolabeled Ag8 bound 2-5% to the CEA immunoadsorbentand 96% to the anti-mouse IgG adsorbent.

Animal Studies. Female hamsters bearing GW-39, a serially propagated, CEA-producing human colonie tumor xenograft (15,16), in both

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RADIOLOCALIZATION OF POLYCLONAL AND MAbs AGAINST CEA

hind leg muscles were used for these studies. In all instances, animalswere transplanted with 0.5 ml of a 10% suspension of GW-39 preparedin 0.85% saline 11-12 days prior to the injection of the radiolabeledantibody. Each radiolabeled MAh was mixed separately with Ag8 sothat groups of 15 animals were injected intracardially with 150-200 ;

RADIOLOCALIZATION OF POLYCLONAL AND MAbs AGAINST CEA

Table 1 Ranking of the tumor localization ability for NP series MAbs and goat antibody 7 days after injectionThe MAbs were ranked according to the means for the percentage of injected dose per g tumor and tumor/nontumor ratios on each day. The example shows the

results from day 7. The /" value (in parentheses) for each successive comparison was used to son the MAbs into groups and a score was assigned to each MAb asdescribed in "Materials and Methods."

Rank%ID°/gtumorPScoreRankTumor/bloodPScoreRankTumor/liverPScoreRankTumor/spleenPScoreRankTumor/kidneyPScoreRankTumor/lungPScoreNP-21.40±0.115NP-22.10±0.134.5NP-28.6

±0.75NP-210.7

±0.95NP-26.3

±0.55NP-24.9

±0.45>

NP-40.65±0.04(0.001)4=

Goat2.10±0.16(0.75)4.5>

NP-46.5±0.6(0.005)3.5>

NP-47.1±0.6(0.001)3>

NP-45.1±0.5(0.05)3>

Goat4.0±0.4(0.05)3.5>

NP-10.44±0.05(0.025)2>

NP-41.60±0.16(0.05)2Goat6.5

±0.6(0.75)3.5NP-16.2

±0.7(0.50)3=

Goat4.6±0.4(0.75)3NP-43.9

±0.4(0.75)3.5=(0.50)_(0.75)>(0.10)_(0.75)=(0.75)>(0.10)NP-30.37

±0.052NP-31.60

±0.152NP-15.1

±0.52Goat5.7

±0.63NP-14.2

±0.53NP-13.0

±0.31.5=(0.75)_(0.50)>(0.05)>(0.001)=(0.25)=(0.75)Goat0.36

±0.062NP-11.40

±0.192NP-33.1

±0.21NP-32.0

±0.21NP-33.4

±0.21NP-32.8

±0.21.5

" ID, injected dose.

2.0 and 2.5, and 3.5 and 4.5, on days 1, 3, and 7, respectively,for the goat antibody, and NP-1, NP-3, and NP-4. For NP-2,

localization indices were 1.8 ±0.3, 3.6 ±0.4, and 5.4 ±0.4 ondays 1, 3, and 7, respectively. Only the localization indices ondays 3 and 7 for NP-2 were significantly higher than the otherantibodies. Thus, all of the antibodies showed a preferentiallocalization for GW-39 in comparison to an irrelevant IgGwithin 3 days.

F(ab')2 fragments were prepared from NP-2 and NP-4 since

these MAbs had the best tumor localization. As shown in Fig.3, the F(ab')2 fragment of NP-2 gave significantly higher tumor/

nontumor ratios than the NP-4 fragment, especially on days 2and 3, despite a similar percentage of each MAb in the tumoron these days. Tumor/nontumor ratios with NP-4 F(ab')2 fragments did not improve as rapidly as NP-2 F(ab')2, and only thetumor/blood and tumor/lung ratios for the NP-4 F(ab')2 sig-

20

15

10

5

0

1.00

0.10

0.01

NP-2 F(ab')2 NP-4 F(ab')2

DAYS POST INJECTIONFig. 3. Distribution of F(ab')2 fragments of NP-2 and NP-4 in hamsters

bearing GW-39 in both hind legs. O, blood; A, spleen; •,liver; A, lungs; D,kidneys; •GW-39.

nificantly improved between days 2 and 3. Thus, the highertumor/nontumor ratios for the NP-2 F(ab')2 were due to the

more rapid clearance of the NP-2 fragments from the bloodand normal tissues in comparison to NP-4 fragments. Thisdifference amounted to almost 10-fold less NP-2 F(ab')2 in theblood than NP-4 F(ab')2, and about 5-fold less NP-2 F(ab')2 in

the normal tissues on day 3. In comparison to intact NP-2 IgG,NP-2 F(ab'>2 fragments had significantly higher tumor/nontumor ratios on all days, but the percentage of NP-2 F(ab')2

fragments in the tumor on day 3 was about 7 times less thanNP-2 IgG, and there was 20 times less radioactivity in the bloodwith the NP-2 F(ab')2. In contrast, by day 3, there was only 2.5times less radioactivity in the tumor with NP-4 F(ab')2 in

comparison to the intact NP-4 IgG, and in the blood, there wasonly about 4 times less radioactivity with the fragment incomparison to the NP-4 whole IgG.

In comparison to the individual MAbs, an equal mixture ofall 4 of the MAbs or an equal mixture of NP-2 and NP-4 didnot improve the localization properties of the antibody (Table2). Although the distribution in the normal tissues was notappreciably altered, the uptake in the tumor was less than theaverage of the means for the individual MAb included in themixtures. For example, when the percentage of injected doseper g tumor was averaged for all of the individual MAbs or forthe combination of NP-2 plus NP-4 on day 7, the percentageof uptake was 0.71 and 1.02, respectively. This is in contrast to0.5%/g tumor found when the antibodies were administered asa mixture. Subsequently, tumor/nontumor ratios generally reflected a value slightly lower than the average ratios calculatedfor the individual MAbs. As shown in Table 2, radiolabelingthe MAbs separately and then preparing the mixture or radio-labeling the MAb as a premixed solution did not influencetumor localization.

DISCUSSION

Our comparison of 4 MAbs that recognize distinct epitopeson CEA has shown that there are statistically significant differ-

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RADIOLOCALIZATIONOF POLYCLONALAND MAbsAGAINSTCEA

Table 2 Biodistribution of the mixtures of the NP series anti-CEA antibodies in comparison to the individual antibodies

% of injected dose/gtissueDays

postinjection137TissueGW-39LiverSpleenKidneyLungsBloodGW-39LiverSpleenKidneyLungsBloodGW-39LiverSpleenKidneyLungsBloodNP-2alone02.3

±0.201.2±0.081.0±0.051.6±0.092.1±0.124.2

±0.222.2

±0.120.5±0.030.3±0.020.5±0.050.7±0.051.5

±0.151.4

±0.110.1±0.010.1

±0.020.2±0.020.2±0.020.6±0.03NP-4

alone1.3

±0.091.0±0.100.8

±0.041.2±0.061.4

±0.084.0±0.381.0

±0.060.3±0.020.3±0.020.5±0.030.6±0.041.3±0.070.7

±0.040.1±0.010.1

±0.010.2±0.020.2±0.020.5±0.05NP

series*

combination0.7

±0.050.7±0.070.9

±0.121.0±0.121.1±0.092.4±0.141.2

±0.090.4±0.030.5±0.050.5±0.060.6±0.061.3+0.140.5

±0.040.1±0.010.2±0.020.1

±0.020.2±0.010.3±0.02NP-seriesc1.4

±0.120.7±0.050.9±0.050.7±0.051.1

±0.092.6±0.160.9

±0.110.3±0.030.4±0.020.3±0.030.5±0.081.1

±0.060.5

±0.040.1±0.010.1±0.010.1±0.010.2

±0.020.4±0.02NP-2

+NP-41*1.3

±0.130.7±0.010.7

±0.100.9±0.131.2±0.103.1±0.151.3

±0.320.3±0.020.3±0.060.5±0.020.5±0.031.0

±0.110.5

±0.060.1±0.010.1±0.010.1±0.010.2

±0.010.3±0.01

" Values represent the means ±SE of the percentage of injected dose per g tissue.* For this combination, 75 Mgeach of NP-1, NP-2, NP-3, and NP-4 were mixed together and then labeled with 5 mCi of "'I. Each animal received 12.5 fig of the

antibody mixture (approximately 3 pg of each M Ab). The number of animals sacrificed on each day (days postinjection) was 6, 9, and 6 on days 1,3, and 7,respectively. Immunoreactivity was 75% on a CEA immunoabsorbent.

' For this combination, NP-1, NP-2, NP-3, and NP-4 were first radiolabeled with 131Iand then mixed together. Each animal received 12.5 ng of the mixture

(approximately 3 ng of each MAh) and 5 animals were sacrificed on each day. The immunoreactivity of each MAh was within 5% of the immunoreactivities reportedin "Materials and Methods" and the mixture's immunoreactivity was 76%.

'NP-2 and NP-4 (75 pg each) was premixed and labeled with '"I and the animals were given injections of 12.5 Mgof the mixture (approximately 6 i/u of eachMAb). A total of 14-18 animals were tested for each day. The immunoreactivity of this mixture was 75%.

enees in the uptake of these MAbs in a human colorectal cellline with less pronounced differences in the distribution amongthe normal tissues. The differences in the tumor uptake of theMAbs could not be explained solely on the affinity of the MAbsfor CEA. As shown in Table 3, NP-2 was ranked third in itsaffinity for CEA by in vitro radioimmunoassay, but ranked firstfor the tumor localization properties. NP-4 ranked the lowestin affinity but was judged to be equal to the goat antibody fortumor localization. Similar results have been found with otheranti-CEA MAbs (10, 11). This suggests that the accessibilityand total number of epitopes expressed within the tumor mayplay a more important role than antibody affinity in governingthe amount of antibody that binds to the tumor. Thus, oneshould not preclude the selection of MAbs for potential clinicalutility solely on the basis of a low affinity binding constant asdetermined by in vitro assays. However, we should view thisconclusion with caution because we do not know the affinityconstants for these antibodies to the CEA that is found in thistumor.

Although statistically significant differences were observedbetween the MAbs in this animal model, tumor/nontumorratios generally were separated by a factor no greater than 1.5.Since the magnitude of these differences was not high, we wouldnot predict on the basis of this study alone that as an intactIgG, any of the MAbs would perform better than the otherswhen given to patients. However, because the F(ab')2 fragments

Table 3 Comparison of the tumor-localizing properties to the affinity constantsfor the polyclonal and monoclonal antibodies

Localizationranking" Affinity constant ranking*

1. NP-22. NP-4, goat3. NP-14. NP-3

1. Goat (1.6 x IO12)2. NP-1 (5.3 x 10")3. NP-2 (1.9 x 10")4. NP-3 (4.5 x 10')5. NP-4 (8.9 x 10')

" Ranking based on the cumulative scores for tumor/nontumor ratios and

percentage of injected dose per gram tumor on each day for all the antibodies.*Affinity constants taken from Primus et al. (12). Expressed as K (M~'(.

of NP-2 gave tumor/nontumor ratios that were not only statistically higher than NP-4, but in magnitude were 3-5 timeshigher, we feel that the fragments of NP-2 may perform betterin patients than NP-4 fragments. Although animal modelsprovide a reproducible means for preclinical screening of MAbs,they may not always predict the precise behavior of the MAbsin patients. Indeed, our clinical experience with NP-2, NP-3,and NP-4 has shown that they all can detect colorectal cancer,but there have been other unique features identified for each ofthe MAbs when they were administered to patients. For example, differences were found in the way the MAbs complexedwith circulating antigen (19), and some reactivity with normalhuman cells was also discovered (20). These clinical findingswill be the subject of a later communication.

In comparison to intact NP-2 IgG, NP-2 F(ab')2 fragments

significantly improved tumor/nontumor ratios on day 3. Incontrast, F(ab')2 fragments of NP-4 gave tumor/nontumor

ratios similar to those of the intact NP-4 IgG on day 3. Theonly exception was the slight improvement in the tumor/bloodratio [0.7 for NP-4 IgG and 1.3 for NP-4 F(ab')2 on day 3].Although NP-4 F(ab')2 was cleared from the blood and normal

tissues more rapidly than NP-4 IgG, it is unclear why thefragmentation of NP-2 resulted in a more accelerated clearanceof the NP-2 F(ab')2 in comparison to the clearance rate of the

NP-4 fragments. Perhaps both NP-2 and NP-4 have some slightreactivity with normal hamster tissues that we have not detectedand the removal of the Fc portion from the MAbs changed theinteraction of NP-2 with the normal tissues more significantlythan that of NP-4. In addition to the relatively slow clearanceof the NP-4 F(ab')2 fragments, our preliminary studies suggest

that fragmentation of NP-4 may have reduced the affinity-binding constant by at least 5-fold (data not shown). Thus,changes in the affinity-binding constant caused by fragmentation may have also contributed to the differences in tumor/nontumor ratios observed for these 2 MAbs.

According to the tumor/nontumor ratios, only NP-2 wassuperior to the affinity-purified goat antibody. Our attempt to

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RADIOLOCALIZATION OF POLYCLONAL AND MAbs AGAINST CEA

simulate a polyclonal antibody by combining the individualMAbs did not improve tumor localization, but instead reducedthe tumor uptake and tumor/nontumor ratios observed withNP-2 alone. Although this observation may be unique to theseMAbs and CEA in this model system, we are unaware of anyother reports that show an improved tumor targeting in vivowith mixtures of MAbs directed against a single antigen. Münzet al. (21) recently reported that a combination of MAbs directed against human colorectal cancer gave higher tumor uptake in comparison to the individual MAbs, as determined byexternal scintigraphic analysis. Although the 2 MAbs used intheir study recognize different antigenic determinants (21), ithas not been reported whether these determinants are found onthe same molecule (22, 23). ( .al far et al. (24) similarly describedan enhancement in tumor/nontumor ratios when polyclonalantibodies against CEA and colon-specific antigen-p were combined in comparison to each antiserum alone. Our resultssuggest that combinations of MAbs directed against multipleepitopes on a single antigen may not enhance tumor uptake,and may even reduce the tumor uptake by reducing the influenceof the best MAb. Theoretically, one would expect a MAbmixture to improve the percentage of uptake in a tumor onlyunder conditions where antigenic sites are saturated by at leastone of the MAbs, are poorly accessible, or under conditionswhere one antibody may alter the expression of the antigen toallow for additional uptake by another MAb. Since we have notbeen able to saturate CEA with NP-2 in this model system (17),nor have we found any augmentation of antibody uptake withthese MAbs, it is not unexpected that we were unable to improvetumor targeting with the MAb mixtures. However, combinations may be useful in order to create a more uniform distribution of the antibodies within the tumor, as in the case wheredifferent epitopes may be expressed at different sites within thesame tumor or if epitopes are expressed differently at metastaticand primary tumor sites. These considerations are importantfor both diagnostic and therapeutic application of antitumorMAbs. Therefore, selection of a MAb or a combination ofMAbs should not only include a consideration of the magnitudeof tumor uptake but also uniformity of its uptake within thetumor.

The tumor/nontumor ratios found with NP-2 are similar tothe results reported by Wahl et al. (25), using their MAb againstCEA in the identical GW-39 hamster-host model. However,since this model is otherwise unique, it is difficult to compareour results to the localization studies reported for other MAbsin nude mice (8, 10, 11, 26, 27). Hence, we are currentlyevaluating the NP series MAbs against a variety of humancolorectal tumors propagated s.c. in the more commonly usednude mouse-human tumor xenograft model. Generally, we havefound a higher percentage of uptake in the GW-39 tumor, adifferent tumor accretion rate, and appreciably higher tumor/nontumor ratios in the nude mouse than in the hamster. However, our studies suggest that at least NP-2 and NP-4 havelocalization properties similar to anti-CEA MAbs reported byothers. Further studies are required to determine whether MAbsagainst CEA or MAbs that react with other colorectal tumor-associated antigens, such as B72.3, (28), 17-1A (27), 79IT/36(29), or colon-specific antigen-p (24), have similar tumor-targeting properties, and if there are advantages to using any oneor combinations of these MAbs.

ACKNOWLEDGMENTS

We thank D. Filion, J. Mabus, and E. Rubin for their excellenttechnical work and L. DePadova, S. Hansen and D. Pawlyk for thepreparation of the manuscript.

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1988;48:1823-1828. Cancer Res Robert M. Sharkey, F. James Primus, Dan Shochat, et al. GW-39 Human Tumor-Hamster Host ModelPolyclonal Antibodies to Carcinoembryonic Antigen in the Comparison of Tumor Targeting of Mouse Monoclonal and Goat

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