radiosensitive tumorssuch as B cell lyinphomas in patients(9,10), they havethe additionaladvantagethat theycanyield precise information concerning biodistribution andtumor dosimetry. These data can be useful for the selectionof the most appropriatetype of antibody for radioimmunoscintigraphy and radioimmunotherapy (9—19).

Monoclonal antibodies used for tumortargetingare generally ofmurine origin and can elicit human anti-mouse IgGantibodies (HAMA) in many patients (20—22).High titersof HAMA are frequentlyobserved afterrepeatedinjectionsof MAbs or when they are coupled to other immunogenicproteins such as toxins or enzymes. Chimerization of anti

bodies representsa first step toward reducingthe immunogenicity of MAbs for applicationin patients (1Z20,23,24).Immunogenicity is, however, still a problem for certain chimericantibodies(25). Furtherhumanizationby graftingonlythe complementaiydeterminantregions(CDR)of the mouseMAb DNA into human IgO DNA as described for the reshaped MAbs (26,27) or production of human antibodies(28-30) might be necessaiy to further reduce immunogemcity.

Chimeric antibodies allow the comparison of the biological behavior of selected human IgO subclasses and verification of their potential for tumor targeting.They can alsobe used to study theireffector functions and the possibilityof obtainingfragmentsfor immunoscintigraphyand radioimmunotherapy (1Z23,31,32). We have used such chimeric anti-CEA MAbs of different IgG subclasses in experimental animal models and could show that both theintact Ig02 MAb and its F(ab')2 fragment demonstratedexcellent tumor targeting and in vivo stability (31). Thetumor localization capacity of the intact Ig04 chimericMAb has been shown to be identical to the origmal mouseMAb in tumor bearing nude mice (23). However, the invivo behavior of chimeric 1g04 F(ab')2 fragments was unsatisfactory in mice (32) and this fragment was thereforenot used in patients.

Here we compare the intact chimeric anti-CEA MAb

Biodistributionand tumoruptakeof a chimerichuman-mousemonoclonalantibody(MAb)andthe originalmouseMAbhavebeencomparativelystudied.Methods: Eighteenpatientswithsuspectedcolorectalcancerscheduledfor surgeryunderwentimmunoscintigraphywith msWabaledthimedcanti-CEAMAb.Iodine-125and 1311trace-labeledchimericandonginalmouseMAb were simultaneousiyinjectedfor b@disthbudonstudies.Results:Similarserumkineticsanda lowimmunogenlcftywereobservedfor bothantibodies.Meanbindingcapadtyto CEAmeasuredinPBSafterradiolabelingwasidenticalforbothMAbsandit wasslightlydecreasedwhenmeasuredin serum1-4 hrafter injection.Radiochromatogramsof patientssera showedimmunecomplexformationrelatedtotheamountofcirculatingCEA.Postoperativeexvivoradioa@tMtycountingintissuesampiesrevealedsimilarantibodydistributionswithnotal@ysimilarantibodyuptakesintumors.Hightumoruptakes(between0.02to 0.06%injecteddoseperg)wereobservedin3 of 13patientsoperatedfor pnmaryor metastaticcokxectalcancer.Concludon: In this dual-labeltechnique,the radiolodinatedanti-CEA19G4chimencMAb and the o@iginaImouse lgG1MAb wereshownto havevery similarbehavlOrin colorectalcancerpatients.

Key Words: chimencmonodonalantibody;carcinoembryonicantigen;tumoruptake;colorectalcarcinoma

J Nuci Med 1995; 36:420-429

he concept of using monoclonal antibodies (MAbs) ascarriers to deliver cytotoxic drugs, radioisotopes or toxinsmore selectively into tumors continues to stimulate experimental and clinical research (1—3).While radiolabeled antibodies have been shown to be useful for therapy of human carcinomas in nude mice (4—8),and of more

RecolvedMay31,1994;revisionacce@*edSept20.1994.Forcorrespondenceandrepdntscont@FranzBuchegger,MD,D@scnof

NudearMedicine.CHIN,CH-1011Lausanne,Switzerland.

420 TheJournalofNudearMedicine•Vol.36•No.3 •March1995

Radiolabeled Chimeric Anti-CEA MonoclonalAntibody Compared with the Original MouseMonoclonal Antibody for Surgically TreatedColorectal CarcinomaFranz Buchegger, Jean-Pierre Mach, AndréPèlegrin,Michel Gullet, Charles-AndréVogel, Thierry Buclin,Jean-Etienne Ryser, Bernard Delaloye, and Angelika Bischof Delaloye

DivL@ionofNuclear Medicine, Depamnentr ofSwgety and Clinical Pharmacolo@, Centre Hospitalier Unive,@itaireVaudoLr and I,t@titute ofBiochemist,y, Univei@y ofLausanne, Lausanne, Switzerland; and L@epa,iment of NuclearMedicine, HOpital Cantonal Universitaire, Genève, Switzerland

(humanI@G4)with its originalmurineIgG@MAb by differential labeling, co-injection and cx vivo measurement oftumor and normal tissue radioactivity distributionsin patients.

METhODS

PatientsPatients selected for the comparativebiodistributionstudy of

chimericand mouse anti-CEAMAb (n = 18)were suspectedofcolorectal cancer. Surgerywas planned 1 to 5 days after MAbinjection. Immunoscintigraphy was performed with the aim ofstaging more precise'y the disease. Definitive diagnosis in thesepatients was: primary colorectal adenocarcinoma in nine patients,

one of whom had initialliver metastasis, one local recurrenceandfive liver metastases. One patient had an ovarian carcinoma, onepatient a benign polyp and one patient with a suspicion of liver

metastasis had no tumorat the time of surgery. For threeof thesepatients, surgely was either performed too late after antibodyinjectionor was cancelled.For determinationof the serum halflifeof chimericMAb,nineadditionalpatientswereincludedwhowere only injectedwith chimericMAb andhada follow-uptimeof2 to 6 days. Finally,one patientincludedherehadsurgeiyforalivermetastasis8 days after injectionof 2 mgof the IgG4chimericMAb together with 30 @gof a chimeric MAb with the samevariableregionsbut with humanIgG2constantdomains(32).

Monoclonal AntibOdiesThemouse-humanchimericmonoclonalantibodyusedhereis

of humanI@G4subclassandwas derivedfromthe murineMAbCE25/B7(6,23)(CIBAGEIGY,Basel,Switzerland).ThisMAbisdirected against the epitope Gold 4 of CEA (33). It has a highspecificity for CEA (34) and does not crossreact with NCA-55 orNCA-95 (35) or other granulocyte glycoproteins. Theoretically,themurineandchimericantibodysubclassesbothhaveminimaleffector functions: they should not react with Fc receptors onmonocytesand macrophagesand shouldnot activatethe complement cascade (36). The original mouse MAb has been used inpatients both for immunoscintigraphy(14) and in a first trial ofrsdioimmunotherapy(1). MouseMAbwas preparedfromascitesby ammonium sulfate precipitation and ion exchange chromatography(6). ChimericMAbwas producedin Sp2@cellstransfectedwitha singlevectorcontainingboththechimericheavyandlightchaingenes (23).

RadlolabelingTwo to 4 mg of chimeric MAb were labeled by the iodogen

methodwith 15 to 30 mCiof ‘@Ifor immunoscintigraphy(finalspecific activities were 2 to 4.5 mCi per mg antibody and 4 to 18mCiwere injectedper patient). Batches of 0.2 mg chimeric MAband oforiginal mouse MAb were separatelylabeled using 250 @.tCiof 1@Iandof ‘@‘i,respectively(finalspecificactivitieswere0.8to 1.1 @&Ciper@ antibodyfor the trace labelings).For threepatients,thetracelabelingswerereversedandthechimericMAbwas labeled with ‘@‘Iand the mouse MAb with 1@I. Using thepairedlabelingmethod,it ispossibleto analyzethebiodistributionand tumor localizationcapacity of the two MAbs in the samepatientand thus compare results obtained underidenticalbiologicalconditions.The 1@I-labeledand the trace-labeledMAbswerepooled, diluted in 100ml of0.9% NaCl and perfused intravenouslywithin15 mm. Total amountof injectedantibody(mouseandchimericMAbtogetheror chimericMAbalone)rangedfrom2 to4 mgin all patients.The15-mmperfusiontime(usedfor safety

reasons) does not significantly influence the pharmacokinetic

analysisbecauseboth antibodieshave been injectedin the sameperfusion and showed a very similar behavior in the hours followinginjection.

T@* Sam@sPatientserumwascollectedimmediatelyfollowingand1,4—6,

24 and 48 hr after injection;additionallyblood samplingwasperformed during surgery (at the moment of tumor removal).Tumorsamplesof primarytumorsor localrecurrenceswereanalyzed together with normal colon and fat. Normal colon tissuewas dissected into the normal mucosa, known to contain CEA(37), and the rest of normalbowel wall. Liver metastaseswereanalyzedtogetherwithlivertissuesurroundingthe tumorandasmall biopsy of distantnormalliver and fat. The tumorwas macroscopicallyseparatedfromnormaltissueand fromnecrosis.Tissue samples were weighed and counted in a triplechannelgammacounter togetherwith a sample ofthe injected material that servedas referencefor the totalinjecteddose. Samplesstillcontaining1231were counted again after complete decay ofthis isotope. Final

radioactivitymeasurementswerecorrectedforcrossoverof 1311inthe 1@Ichannel.

In Vitro Testing of Radlolabeled MAbsTheinvitroimmunoreactivefractionof radiolabeledchimeric

and original mouse MAb was determined in a binding assay onCEA insolubilizedon CNBr-Sepharose(Pharmacia,Uppsala,Sweden).Tento 50 nCiof radiolabeledMAbwereincubatedfor16hrat 25°Cin PBSbuffercontaining1%normalmouseserumand1%normalhumanserumwith5 @dpackedCEA-Sepharose(containingabout2 @&gpurifiedCEA).Afterwashing,boundradioactivity was determined as percent of input radioactivity. Similarly, patients, serum samples obtained 1 to 4 hr after injection(containingsimilar amounts of radioactivity)were diluted 113inPBS buffercontaining1%normalmouseserumandwere alsoincubatedwith 5 @1packedCEA-Sepharose.Nonspecificbindingwas measured by incubation with irrelevant protein also coupledto CNBr-Sepharose.Itwas alwaysbelow2%andwas subtractedfromCEAbindingvalues. Trichloroaceticacid precipitationofradioactivityafterlabelingshowedthat morethan95%of theradioactivity was bound to protein for all preparations. MalytiCaIsize chromatographyof the radiolabeledMAbs was done on aSephadexG-200columnor on a Superdex200 FPLCcolumn(bothPharmacia).Immediatelyafterlabeling,a sharppeakwasobtainedforbothMAbswithoutdetectableamountsof aggregatesandwithonlytraceamountsof freeiodine.

CEA and HAMA AssayCirculating CPA was determined in a serum sample of each

patient taken before injection of radiolabeledantibodies using apreviouslydescribedsolidphase enzymeimmunoassay(38).

HAMA and anti-idiotype antibodies were measured in threesandwichassays.Briefly,forthethreetestsA, B andC, polystyreneballswerecoatedwithA), irrelevantmouseIgG,B), mouseMAb CE 25 or C), chimenc MAb, respectively. These balls wereincubated for 3 hr at 25°Cwith 10 p1 patients serum (taken 5—6wkafter antibody injection)diluted in 300 @lPBS and peroxidasecoupledmouseMAbCE25(testA andB)orradiolabeledchimericMAb in test C. A rabbit anti-mouse F(ab')2 antiserum (10 @.dserumdiluted1:10'000in PBS) servedas a positivecontrol.Normalhumanseraservedas negativecontrols.All patientsseratestedbefore any MAbinjectionwere also negative.

421@himencversusMouseMAbin Patients•Bucheggerat Si.

Patientno.Age SexDiagnosisDifferentIatiOnDukesSurgery(day)147

MPrimaryrectumIntermB2271FUver met.sigmoldIntermC1369FPrimary cciascIntermB2478FPrim andmetcci

ascIntermD1570FOvarlanadenocaLow5657

MHyperplasticPolyp1765FLocal rec.sigmoldIntermC2879MPrimarysigmoldInterm-wellBI944

MPrlmatysigmoidLowC21073FPrimarysigmoidIntermB—1

187 MPrimarysigmoidmt-wellD11266MPrimaiycoltrIntermB—1364

MUver metsigmokinaB—1463MLiver metrectumIntern,C21

574 MLiver metcolonnaC21672FLiver metrectumIntermC51742FSuspected liver

mata@asin11858 FPrimary colondrIntermB2na

=not available.

TABLE IPatientsInjectedwithChirnedcandOnginalMouseAnti-CEA

MAbsandMajorClinicalParametersPharmacokineticswere analyzedby modelinga time (hr)—

radioactivity (cpm/ml) curve for each patient. Time 0 immediatelyafter injection was taken as 100%.Using the SIPHAR program(Simed,Creteil,France)individualpatientdatawereanalyzedinatwo-compartmentmodel.Aweightedleast-squaresmethodwithweights being the reciprocal of the predicted radioactivity wasusedto estimatetheparameters.A linearcorrelationanalysiswasusedto calculatethecorrelationcoefficientbetweenthechimericand the original mouse MAb in serum.

RESULTS

Eighteen patients were injected with ‘@I-labeledchimeric anti-CEA MAb together with “SIand ‘@‘Itracelabeled chimeric and original mouse MAb (Table 1). Although immunoscintigraphy is not the objective of thisstudy, two representative illustrations of a primaiy and ametastatic tumorimmunoscintigraphyare shown in Figure1.SerumCEAof thesetwo patientswaslow (1.7and1.3ng/ml) and immune complex formation was almost negative. Both immunoscintigraphsclearly show antibody uptake in the tumor 6 and 24 hr after injection. At these times,blood radioactivity was still high as it has been observedearlier after injection of radioiodinatedintact MAbs.

B-:@‘@

A

FiGURE 1. (A)Uptakeof thimeiic anti-CEAMAbIna primarytumorof the colonascendens(Patient3) and (B)in a livermetastasisofacoloncarcinoma(Patient19).InPanelA,a6-gprimarytumorofthecolonascendensiscleedyvisibleontheplanarview6hrafter1@l-MAbinjection.PanelBshowsa coronalsectionoftheabdomenofa patientwithan8-9livermetastasisintheleftliverlobe24hrafter1@IMAbinjection.Uptakeinthe metastasis(arrow)is ofsimilarintensitythan that of heartand blgvessela,witha hypoactivecentercorrespondingtonecrotictissue.Activityinnormalliverislow.

422 TheJournalofNuclearMedicine•Vol.36•No.3 •March1995

100

•0•apg60ga

@40@ :-‘•U0 20

hours aft.r Injic010n pircantB/

400080100dcr•aa•chimiric

MAb

A

(

300Ca@Wb

ic.i:C —..— chi..•.@I

@,1500

@ 1000U

500

020 30 40 50 60 70

CI IIIj@@*30

40 50 60 7030 40 50 6070tubs

numbar

FIGURE2. (@4@Radk,actMtyconcentrationsof thechimericMAb(0) andof the originSimouseMAb4). Arrowslnd@atepercentInsaraof Patients2 and4 andshowexceptionallyshortcirculationtimesfor bothchimerleMAbandoriginalmouseMAb.(B)Serumdisappearanceof mouseMAbIsplottedaginstthstofct@medcMAbofallserumsam—InPanelA(Patients2and4arenot—. Theca@stedregressionsWSig@linehasthecharactedstlcsof

Y=-1.54+1.146x;correlationcoefficientr@= 0.95.

Overall,asimilarserumdisappearancekhietlcofbothantibodyformsisapparentwltha marginallyshorterhat-lifeforthemouseMAb.

Sirum KineticsThe serum half-Me of the two MAb forms was very

similar in all patients with a tendency of marginallylongerretentionof the chimeric MAb laterafter injection(after24to 48 hr, Fig. 2A).

Many of these patients were followed for only one totwo days since serum collection was discontinued aftersurgery. Frequently, this short observation time did not

permit us to obtain a clear result concerning serum alphaand beta half-lives. It appears, however, from the individual data shown in Figure 2A that there is only a minordifference in the circulationkinetics between the two antibodies. Thus, a high correlation (correlation coefficient r@= 0.95) was observed for the two radiolabeled antibody

fonns in the serum samples as shown in a double plotanalysis (Fig. 2B).

In two patients (Patients2 and 4), the serum half-lives ofboth the chimeric MAb and of the original mouse MAbwere very short (5 and 7.1 hr) and only between 6 to 13%of radioactivity remained in circulation after one day forboth antibody forms. In one of these two patients (Patient4) the accelerated half-lifewas most likely due to bindingofthe antibodies to CEA, since no HAMA and no anti-idiotypes were detected in his serum. Indeed, he had a highamount of serum CPA (342ng/ml) and 47% of the chimericMAlIand 43%of the mouse MAt, were in aggregatedformearly after injection, as determined by size chromatography on Sephadex 0200 (Fig. 3C). For the second patient(Patient 2), no obvious reason for the short half-lifeof thetwo antibody forms was found. CEA in serum was relatively low (24 ng/ml), and both antibody forms appeared tocirculate at a high percent in monomeric form: the serumcollected4 hr after injectioncontainedonlyabout4%ofaggregates as determined by Sephadex G200 radiochromatography. No indication of increased dehalogenation inserum was found since only low amounts of free iodine(

PedantPercentbindingCEAinChimericMAbMouseMAbno.In

vitroin serumIn vitroIn serum(nglml)

TABLE 2Percent Bindingof the Two RadiolabeledMAbs Before and

AfterInjectionandSerumCEALevelsEaa

C

Ua0

VS

CaU

a0.

1 50 52 61 52 25.22 82* 80 50 60 23.83 76 73 80 75 1.74 53 27 76 35 3425 53 32 76 35 0.36 53 48 76 52 1.37 75 28 76 30 1378 79 43 56 31 2.89 80@ 35 70 36 16.7

10 81 61 72 56 3311 81 18 79 20 26.712 81 16 79 19 9.613 76 42 76 46 3314 84 75 82 84 1.915 83 81 81 80 9016 77* 63 67 63 2.017 68 74 71 69 0.918 63 51 60 59 nd.

amthreepatients,tracelabelingofchimericMAbandoriginalmouseMAbwaswith1311and1@l,respectively,Insteedof the normallyused‘25t-1al@eledchimericMAband‘@‘l-labeledodginalmouseMAb(reversicnof Isotopes).

nd = notdone

of circulating CEA in whom about 2%, 10% and 45% ofMAb aggregates were found. Note the appearance of significant amounts of free iodine (5% to 6%) only in theserum of Patient 4 (Fig. 3C) who had more than 40% ofimmune complexes.

HAMA were tested in 9 patients 5 to 6 weeks afterinjection of chimeric and mouse MAb. One patient developed anti-idiotype antibodies reacting with the chimericmolecule as well as HAMA to irrelevant mouse immunglobulin. An additional patient (mentioned above) hadHAMA reacting only with the mouse immunglobulinbutno reactivity against the chimeric MAb was detected.

Antibody Blodistributlon In Tumor and Normal TissuesFifteen of the 18 patients injected with both antibody

forms underwent surgery 1 to 5 days after injection (Table

1). In one patient, a suspected malignantrecurrenceturnedout to be negative (Patient 17) and in another patient abenign polyp was removed at surgery while a first polypshowing malignant transformation had been completely removed duringendoscopy before injection of the antibody(Patient 6). In a third patient with suspected adenocarcinoma of the rectum, the final diagnosis was a non-CEAproducing ovarian carcinoma infiltrating the rectum(Patient 5).

Primary adenocarcinoma of colorectal origin or a locoregional recurrence were surgically removed from eight patients includingboth patients with short serum T1,@of the

24 48 72 96 120hours aftar injection

FiGURE 4. Serumradioactivityof chimericMAbfor 12 patientsfollowedfor2to6days(dottedlines).Thethickcurverepresentsthemedianhalt-lifecalculatedfor a two-compartmentmodelwith thecharecteristicsof

medIanTi,@a7.2hrandmedianTiap91.1 hr,

leadingto:

Y = (lOON) - (0.685 - e@1t+ 0.315 . e@),

whereV = 0.99,A1= 0.0076and A@= 0.097.In addition,the t@thinstra@itlinesrepresentthecalculatedmedianalphaandbetahalt-lives(the beta halt-lifestraightIkie Is fused with the medianhalt-lifeafter48 hr).

ies. There was no direct correlationbetween the decreaseof antibodybindingto CEA andthe percentageof antibodyaggregates. Interestingly, except for Patient 6, all otherpatients had a similar decrease of CEA binding for thechimeric and mouse MAb. Overall, binding capacity inserum was decreased as compared to binding after labelingby about 22%for both the chimeric MAb and the originalmouse MAb. The mean bindingof chimeric MAb in bufferand in serum was 71.9 ±12.0 and 50 ±21.3, respectively.The corresponding figures for mouse MAb are 71.6 ±9.2and 50.1 ±19.9.

Analytic size chromatography was performed on thefreshly labeled MAbs and on the early serum samples,which allowed the % of in vivo aggregatedantibody to becalculated. The formation of immune complexes correlatedwith the amount of circulating CEA. Taking arbitrarily30ngof circulatingCEA/m1asthelimit, 12of 17patientshad lower serum CEA levels and the percentage of awegates was 3% to 14%.In contrast, all 5 remainingpatientswith CEA levels higher than 30 ng/ml had more than 15%of aggregates for both the chimeric MAb and the mouseMAb. In one patient with 342 ng/ml of circulating CEA(Patient 4), 47% and 43% of injected chimeric and mouseantibody, respectively, were found as immune complexes.At the time of antibody injection, none of these 5 patientshad significantHAMA titers. In one of them (Patient 10) alow titer of anti-mouse IgU antibodies (test A + B) wasfound five weeks after injection, but no reactivity againstthe chimeric MAb appeared.Figure3 shows the radiochromatograms of three representative patients with low (2.8ng/ml), medium (26.7 ng/ml) and high (342 ng/inl) amounts

424 TheJournalof NudearMedicine@ Vol.36@ No.3@ March1995

PatientnumberTumorNormalbowel

waitChimericMAbMouse MAbChimeric MAbMouse MAb

I

IILI

!

SpM$int2SaI:

I:@

@ I I ‘.11

*MeasuredseparatelyasshownInFigures5-7.@Tumorand normal bowel wait redloactMtyuptake is expressedIn

%ID/gtlssuex*Tumor@tD@nonnalbowelwallredloactMtyratiosaregh,enInparen

theses

I

I

I@I!E@;i@ !,gI@

;@@ ! @‘

!

TABLE 3Comparisonof Tumor Uptakeof RadiolabeledChimericandOr@nSiMouseMAbto NormalBowelWallSthppedfrom

NormSiMucosa*

I9.8@10.51 .5 (6.5)@I.5(7.0)37.97.93.4(2.3)3.1(2.5)42.03.11

.0(2.0)0.9(3.4)710.111.04.7 (2.1)3.1(3.5)814.29.42.1

(6.8)1.7(5.5)98.08.21.6(5.0)1.7(5.0)1116.917.82.3(7.3)1.7(10.4)1852.655.73.6

(14.6)2.2(25.3)FIGURE 6. Tumorand normaltissue blodlstñbutionof chimericanti-CEAMAb IgG4(darkshading)and od@nSimouseMAb (lightshading)intwopatientswhohadsurgeryforIniermetastasesofthecolonand rectalcardnorna,respectivaly.Verticalbars indicatetherangefor two or morepieces.Patient4 hed surgeryfor a primarytumor and a liver metastasis.Uptakeof both MAbs In the latterexceededthatoftheprimarytumor,whereasuptakeInnecroticpartsof the livermetastasiswas not higherthan that of adjacentnormalliver.

antibodies. In these eight patients the mean % ID/g tumorwas identical for both MAb with 0015% and a large scatter, while the percentages in the normal bowel wall,stripped of the CEA producing mucosa, were 0.0025% and0.002% for the chimeric and the mouse MAb, respectively(Table 3). Figure 5 shows one patientwith a rectum carcinoma (Patient 1) with a typical median antibody biodistribution and in comparison the result of the patient with anovarian carcinoma (Patient 5). In this second patient, theantibody concentrations in the ovarian malignancy (without evidence for productionof CEA) was much less thaninblood and also less than in the normal bowel wall. Figure 6shows an additionalpatient with a colon carcinoma(Patient 4) having a very low uptake in its primarytumor(probablyrelatedto highamountsof CEA in the circulationand immuncomplex formation), while Figure 7 shows apatient with a primary colon carcinoma and a very highantibody uptake (Patient 18).

Seven liver metastases were surgically removed andcounted from patients after injection of both the chimericMAb Ig04 and the originalmouse MAb. One patient presented with a primarytumorand a liver metastasis (DukesD, Patient 4), Patient 2 had a large liver metastasis and amicrometastasis and Patient 16 had two liver metastasesthat were surgically removed and analyzed.

In the seven metastases, for the chimeric and the mouseMAb, the mean % ID/g tumor was 0.015 and 0.014%,respectively, with a large scatter, while the percentages innormal distant liver were 0.0017% and 0.0023%, respectively (Table 4). Figure 6 shows one patient (Patient 16)who had two liver metastases from a colon carcinomawith

FIGURE 7. Tumorand normaltissueblodistributlonof chimericant@CEAMAb IgG4(darkshacting)and or@nSimouseMAb (lightsheding)Intwopatientswhohal surgeryfor primarycoloncardnomaandlivermetastaSis.VerticalbarsIndicatetherangefortwoormore pieces.In Patient18,a high uptakeof bothantibodieswasobservedin the primarytumor.The lymphnodewas foundtumorfree by histologicexamk@ation.In Patient2, a highconcentrationofbothantibodieswas foundIn a 60-mgmicrometastaals,whileantibodyuptakein a 76-9metastasiswasmuchless.Themicrometastasisshowedfive to six times higherantibodyuptakethan thelargertumor.

FiGURE 5. Tumorand normaltissuebiOdiatIlbutiOnof chimedcantl-CEAMAb 1gG4(darkshading)and originalmouseMAb (lightsha@ng) in two patients (one with rectal and one with ovarian carcinoma).Verticalbars indicatethe rangefor tissueswheretwo ormore pieceswere available.Clear uptake is shown In the rectalcarcinomaof PatientI, whereasno uptakeia demonstratedin theovariancarcinomaof Patient5.

ChimericversusMouseMAbinPatients@ ButheggeratSi. 425

.. S !@

S@@

it t I Iit j ft

Patientno.Liver

metastasisNormalliverChimeric

MAbMouse MAbChimeric MAbMouse MAb

•Radk@actMtyuptake In the liver metastasIsand normal liver is expressedin %lDIgtissuex

tp@@ comparinglivermetastasisradloactMtyto that In normalliveraregivenInparentheses.

*AntIbodyuptakeina micrometastasis0140mgfoundduringdissectionof normallivertissueadjacentto thelargemetastasis.

a typical mean antibody uptake in tumor. A second patientshown in Figure 6 (Patient 4) presented with a colon carcinoma and with an initial liver metastasis. While antibodyuptake in both tumor sites was low, uptake in the livermetastasis was about two times higherthan in the primarytumor. This patient had the highest amount of circulatingCEA and aggregated antibodies in the serum (43% and47%) and a very short half-life of both antibody forms thatmight explain the low uptake of antibodies in the tumors.Patient 2 shown in Figure 7 was operated for a liver metastasis 1 day after antibody injection. While the largetumor (75 g) had a mean uptake of about 0.010% ID/g, amicrometastasis of 40 mg, found at dissection of the adjacent normal liver tissue, had an uptake of 0.062% and0.038%injected dose per g for the chimeric and the mouseMAb, respectively, in other words, five to six times higherthan in the large metastasis.

A finalpatient is presented in Figure8 who was operatedfor a liver metastasis 8 days after injection of the Ig04chimeric MAb labeled with 1@I, together with a smallamount (30 /Lg)of the Ig02 chimeric MAb, labeled with1311 In this patient, 3.5 to 4.5 times higher tumor-to-blood

and tumor-to-liver ratios were obtained with the chimericMAb of IgG2 subclass than with the IgG4 chimeric MAb.Both antibodies injected in this patient (having the samevariable domains) showed highbindingto CEA afterradiolabeling (79%for the Ig04 chimeric MAb and 88%for theIg02 chimeric MAb) that was only marginally decreased inserum. Ifconfirmed the surprisingly higher tumor uptake ofthe IgG2chimeric MAb would suggest its use in patientsinstead of the Ig04 chimeric MAb.

DISCUSSION

In this study eighteen patients were injected with achimeric MAb of human IgG4 subclass together with theoriginal mouse 1g01 MAb. Both are directed against thesame epitope ofCEA and have an identical affinity(23,32).

TABLE 4Comparisonof RadiOlabSiedChimencand OnginSiMouseMAb Uptake in Uver Metastasesand Normal Uver Thsue

— — patI.nt 19 •@@

@ 0 c@suss

i:11111@i@j1TTT

210.4*7.82.1 (5.O)t4.8(1.6)262.6@38.12.1(29.8)4.8(7.9)44.37.31

.2 (2.7)2.1(3.5)148.517.61.3(6.5)1.3(13.5)154.63.81.8

(2.6)1.7(2.2)166.49.32.1(3.0)1.4(6.6)168.212.12.1

(3.9)1.4(8.6)

FiGURE 8. Comparisonof tumor uptakeof chimencIgG4MAb(darkshading)withthatof IgG@subclass(openbars)that has identicalspecificity(becauseIt wasdeducedfromthe sameoriginalmouseMAb) In a patientwfth liver metastasisfrom coloncancer.The livermetastasiswassurgicallyresected8 daysafterInjectionofthetwotrace-labeledantibodies.Verticalbarsindicatethe rangefort@ or morepiaces.

The capacity of antibodies to localize in tumors dependson multipleparameters,amongothers on the immunoreactive fraction after radiolabelingand injection. Tumor size,the interstitialfluidpressure (40), vascularization and vascularpermeability(41) tightjunctions in well-differentiatedtumors and the so called binding-site barrier (42) (absorption of high-affinityantibodies to antigenon tumorsurface)can further modulate antibody uptake. The comparison oftwo antibodies using the double-labeling technique,whereby both antibody forms encounter identical biological parameters in each patient, allows a meaningful comparativeanalysis in a limitednumberof patients. Since theoverall total amount of antibody injected in this study(4 mg) was low, tumor antigen was in excess and no majorcompetition for antigenic sites should have occurred.

Overall, tumor uptakes and normal tissue radioactivitydistributions showed a similar behavior for both MAbforms. Some variationwas observed in individualpatientsthat can be attributedto a certain degree of variability inthe different batches of MAb preparations and in iodinequality and labeling. The in vitro quality controls reflectpart of these variations while others might be apparent onlyin vivo as suggested by other studies (3Z39).

In serum, the chimeric MAb had a marginally longerhalf-Me than the original mouse MAb. This was observedwhether patients had high or low amounts of circulatingCEA. Since both antibodies have an identical affinity forantigen (23,32), the percentage of immune complexes forboth was very similar in individual patients related to serum CEA. Apparently, the elimination of these immunecomplexes by the RES was also similar. The measuredhalf-life of the IgG4 chilneric MAb was shorter than expected. It is possible thateven small amountsof circulatingCEA induced formation of immune complexes that influenced the beta half-life. Additionally, the observation time

426 The JoumSiof Nudear Medicine@ Vol. 36@ No. 3@ March 1995

is relatively short in many patients and might have influenced evaluation of this half-life in some patients.

In the present comparison of mouse and chimeric MAb,we measured the immunoreactivefractionbefore and afterinjection. While the sera of certain patients had little or noinfluence on the binding capacity of the antibodies, in otherpatients the binding capacity of both of them was drastically reduced afterinjection. Reducedbindingwas found inpatients with large amounts of immune complexes due tocirculatingCEA, as in Patients 4, 7 and 13. In some cases,however, reduced antibody binding after injection wasfound in patientswith low serum CEA, as in Patients5 and8. In these patients, the diminished binding after injectionmight be due to a non-specific influence of serum on antigen-antibodyassociation: ionic strengthhas been shown toinfluence the affinity of antibodies against a closely relatedepitope ofCEA by a factorofup to 100(43). Ourassay thatmeasures bindingof radiolabeledantibody to a limited cxcess of antigenmightbe particularlysensitive to such nonspecific binding inhibition in serum.

Our results concerning HAMA and anti-idiotype antibody formationin patients show that both the radiolabeledchimeric and original mouse MAb have a low immunogenicity after a single injection. Several other reports haveshown a higher immunogenicity of mouse MAbs, especially after repeated injections, but also low immunogenicity after single injections of MAb have been reported (20—22). Our results are in line with our previous observationswhere after a single injection of radioiodinated mouseMAbs for immunoscintigraphy detectable HAMA titerswere only rarely found. Such a first MAb injection might,however, stimulate formation of memory cells in manypatients. This is suggested by the fact that a second MAbinjectionwas followed frequentlyby a rapidappearanceofimportant HAMA titers already after 2 wk (unpublishedobservation).

For chimeric MAbs, a weak immunogenicity has beendescribed more frequently (12,24). However, one studywith the chimeric human IgG4 MAb B72.3 showed that ithad a high immunogenicity (25): after a single injection of3.4 to 6.9 mg, 7 of 12 patients developed measurableHAMA titers against this antibody (25). This MAb had alongerserumbeta half-life(224 ±66 hr)as comparedto ourchimeric anti-CEA MAb (91hr median beta half-life) and isdirected against an antigen that is less frequently elevatedin patient's serum. Since the two MAbs have differenthuman Ig64 constant domains, it remains unclear whetherthe higher immunogemcity of the B72.3 IgG4 chimericMAb is due to its different mouse variable domains or tothe differentallotypic epitopes on the human constant domains. Interestingly, the formation of immune complexesin our patients does not appearto promote HAMA formation. Immune complexes are preferentially bound by thelow affinity IgG Fc receptors on macrophages (44) andhave been used in immunizationprotocols.

Of interest are a few data concerning high tumoruptakeof radiolabeled MAbs: Figure 7 shows a very good local

ization for both the chimeric MAb and the original mouseMAb in a primary tumor operated two days after antibodyinjection (Patient 18). Similarhigh antibody uptake in primary tumors has been observed in a series of six patientswho were injected with higher amounts of the same chimeric anti-CEA antibody (10 mg per patient) trace labeledwith radioiodineand coinjected with the identical antibodycoupled to fluorescein for the purpose of immunophotodetection (45). Figure 7 shows another patient operated for aliver metastasis where on dissection of the adjacent normalliver tissue, a micrometastasis of 40 mg was found (Patient2). Here, the micrometastasis showed, on a per gram basis,a five to six times higher antibody uptake than the largemetastasis. This patient had a very short circulation halflife for both antibody forms that remains unexplained. Patient 19, who had surgery eight days after antibody injection, had a relatively high tumor uptake as compared tonormaltissues: 0.01%of the injected dose per g tumorwasfound for the Ig04 chimeric MAb, and more than 0.02%ofthe coinjected Ig62 chimeric MAb labeled with 1311.Thetwo chimeric MAbs of different human IgO subclasses arederived from the same anti-CEA mouse MAb and have asimilar affinity for CPA (32). Interestingly, the chimeric1g02 MAb injected in trace amounts (30 @g)gave 3.5 to 4.5times highertumor-to-liverand tumor-to-bloodratios thanthe Ig04 Chllfl@riCMAb. While the limited amount of antibody injected might explain the faster disappearancefromblood for the chimeric IgG2 MAb@its higher tumor uptakecertainly merits further investigation.

The three patients mentioned above with particularlyhigh antibody uptake in tumor stimulate some reflectionsconcerning dosimetry for a potential radioimmunotherapy.Consideringradioimmunotherapyin a postoperative adjuvant setting where occult micrometastases would be thetarget, such micrometastases might accumulate higher antibody doses than large tumormasses as it is suggested bythe results from Patient 2 and by other reports (7,17,46).Thus, a tumor uptake in the range of 0.06% ID/g could beobtained more frequently in small tumors as compared tolarger ones. Assuming homogenous irradiation of suchsmallnodules with a radionucide emittingbeta-radiationofmedium energy (e.g., 1311or 67@), the MIRD formulawould predict an irradiationof 2100 rad for an injection of100 mCi ‘@‘Iand a 60-hr effective half-life in the tumor. Fora repeated injection of 2 x 100 mCi, the total tumor dosewould reach 4200 rad. Such radiationdoses in micronodules thatareoptimallyoxigenated andradiosensitivewouldhave a good chance to be efficient. The effective tumorradiationdose could, however, be modulated by parameters such as the percent of absorbed energy in a givenmicronodule and for a given radiation type (47), the microheterogeneity of tumor irradiationdue to uneven distribution of antibody (48) and the low dose rate of irradiationthat is generallyobtainedin radioimmunotherapy(49). Theextrapolationof our results and those fromother groups asshown above should, however, encourage future experi

427chimencversusM@ i@i@in Patients@ Bucheggerat Si.

mentalandclinical radioimmunotherapystudies of minimalresidual disease.

Altogether, the results obtained with the chimeric antiCEA IgG4MAb showed a tumor localization capacity thatis comparableto that of the originalmouse MAb which hadbeen selected for high affinity and antigen binding. Thetumor uptake of these two MAbs was similar or evenslightly superior to that observed with anti-B cell MAbs(9,10) which have been successfully used for radioimmunotherapy of lymphomas.

ACKNOWLEDGMENTS

The authorsthankMrs. K. Foamier for efficienttechnicalassistance. We are grateful to Dr. F. Healy for reviewingthemanuscript.The chimericMAb wasproducedby CibaGeigy,BaselSwitzerland.Thisworkwassupportedby theSwissFoundationfor ScientificResearch(grantnumber31-31238-91),byRechercheSuisse contre le Cancer(Akt 312) and the RobertWenner Swiss Cancer Research Award 1991.

REFERENCES1. MachJ-P,PulegrinA, BucheggerF. Imagingandtherapywith monoclonal

antibodiesin non-hematopoictictumors.CurrOpEnImmwsol1991;3:685—693.

2. Scheinberg DA. Current applications ofmonoclonal antibodies for the therapy of hematopoleticcancers. Cwr OpinImmwsd 1991;3:679-684.

3. OoldenbergDM,Schiom3. Thecomingof ageofcancerradloimmunoconjugates. Immunol Today 1993;14:5-7.

4. CheungNK, LandmeierB, Neely3, et at.Completetumorablationwithiodine131-radiolabeleddiSialOgangliOsideGD2-specilicmonoclonalantibody against human neuroblastoma xenografted in nude mice.JNatI Canca@Inst 1986;77:739—745.

5. SenekowitschR, ReidelG,MOlleUStãdtS, KriegelH, PabstHW.Curativeradioimmunotherapyof humanbreasttumorswith 131-I-labeledmonoclonat antibodies. JNuclMed 198930:531-537.

6. Buthegger F, Pfister C, Fournier K, et al. Ablation of human colon cardnoma in nude miceby 131-I-labeledmonoclonalanticarcinoembiyonkantigenantilxxlyF(ab')2 fragments.I ClinInvest 1989;83:1449-1456.

7. Buchegger F, PèlegrinA, Delalcye B, BischofDelalcye A, Mach J-P. 131-Ilabeled F(ab')2 fragments are more efficient and less toxic than intact antiCEA antibodiesin radioimmunotherapyof large human colon carcinomagraftedinnudemice.JNucIMed1990;31:1035—1044.

8. Sharkey RM, Weadock KS, Natale A, Ct al. Successful radioimmunotherapy for lung metastasis of human colonic cancer in nude mice. I NailCancerlnst 1991;83:627—632.

9. Press OW, Eary iF, Appelbaum FR, Ctat. Radiolabeled-antibody therapyofB-celllymphomawithautologousbonemarrowsupportNEnglJMed1993;329:1219—1224.

10.KaminakiMS, ZasadnyKR, FrancisIR, et aL RadioimmunotherapyofB-celllymphomawith (‘311)anti-B1(anti-CD2O)antibody.N E@ogII Med1993329:459-465.

11. Breitz HB, Weiden PL, Vanderheyden J-L, Ctal. Clinical experience withRhenium-186-labeledmonoclonal antthodies for radioimmunotherapy: resuIts of phase I trials. JNucl Med 1992;33:1099-1112.

12. LoBugJio AF, Wheeler RH, Trang J, Ctal. Mouse/human chimeric monodonal antil,ody in man: Kinetics and immune response.PmcNadAcadSciUSA 1989;86:4220—4224.

13. Delalcye B, BischofDelalcye A, Buchegger F, Ctal. Detection of colorectalcarcinomaby emission-computerizedtomographyafter injectionof 123-I-labeledFabor F(ab')2fragmentsframmonoclonalanti-carcinoembiyonicantigen antibodies. I Clin Invest 1986;77:301-311.

14. Bischof Delaloye A, Delalcyc B, Buchegger F, Ct al. Qinical value ofimmunoscintigraphyin colorectalcarcinomapatients:a prospectivestudy.JNucI Med 1989;30:1646-1656.

15. RyserJE,JonesR, Egei R, et al. Coloncarcinomaimmunoscintigraphybymonodlonalanti-CEAantilxxlylabeledwith gallium-67aminooxyacetyldeferroxamine. INuciMed 199233:1766-lm.

16. Begent RHJ, Ledermann JA, Green Al, et al. AntilXXIydistribution and

dosimetiyinpatientsreceivingradiolabelledantilxxlytherapyforcolorectalcancer.BrI Cancer 1989;60:406-412.

17. @haadJ-F, Saccavini J-C, Gestin i-F, Ctat. Biodistribution of indium-illlabeled 0C125 monoclonal antibody intraperitoneally injected into patientsoperatedon for ovariancarcinomas.CancerRes 1989;49:3087—3094.

18. MilenicDE, Yokota1, FilpulaDR,et al. Construction,bindingproperties,metabolism,andtumortargetingof a single-chainFv derivedfromthepancarcinomamonoclonalantibodyCC49. CancerRes 1991;51:6363-6371.

19. Buchsbaum Di, Weasels BW. Radiolabeled antibody tumor dosimetiy.MedicalPhys 1993;20:499—501.

20. Kuus-ReichelK, GrauerLS,KaravodinLM, KnottC,KrusemeierM, KayNE. Minireview.Will immunogenicitylimittheuse,efficacy,andfuturedevelopment oftherapeutic monoclonal antilxxlies? Clin Diagn Lab Immunol 1994;l:365—372.

21. HyamsD, ReynoldsJC, CarrasquilloJA, et al. The effectof circulatinganti-murineantthody on the pharmacokineticsand biodistributionof injected radiolabeled monoclonal antthody [Abstracti. I Nuci Med 1986;27:92@

22. Abdel-NabiHH, Doerr Ri, Chan H.W, et aL Safety and role of repeatedadministrationof Indium-ill-labeled anticarcinoembiyonicantigenmonoclonal antilxxly ZCE 025 in the postoperative follow-up ofcolorectal cardnoma patients.JNucIMed 199233:14-22.

23. HardmanN, Gill LL, De Winter RF, et al. Generationof a recombinantmouse-human chimaeric monoclonal antibody directed against human carcinoemblyosicantigen.Intl Cancer 1989;44:424-433.

24. GoodmanGE, HellstrOmI, YeltonDE, et al PhaseI trialof chimeric(human-mouse)monoclonalantibody L6 in patients with non-small-celllung,colon, and breast cancer. Cancerlmmwzollmmunother1993;36:267-273.

25. Khazaei MB, Saleh MN, Liu TP, et al. Pharmacokineticsand immuneresponseof131I-Qtimencmouse/humanB72.3(humangamma4)monoclonal antibody in humans. CancerRes l991@1:5461-5466.

26. RiechmannL, Qark M, WaldmannH, Winter 0. Reshapinghuman antibodiesfortherapy.Nature1988;332:323—327.

27. SingerII, KawkaDW,DeMartinOiA, et al. Optimalhumanizationof 1B4,an anti-CD18murine monodonal antil,ody, is achieved by correct choice ofhumanV-regionframeworksequences.Jlmmunol 1993;150:2844-2857.

28. GriffithsAD,WilliamsSC, Hartley0, et al. Isolationoflugh affinityhumanantthodiesdirectlyfromlargesyntheticrepertoires.EMBO! 1994;13:3245—3260.

29. Lonberg N, Taylor LD, HardingFA, et al. Antigen-specifichuman antibodiesfrommicecomprisingfourdistinctgeneticmodifications.Natw@1994368:856—859.

30. MorrisonSL Successinspecification.Nature1994;368:812-813.31. VogelCA, BischofDelaloyeA, MachJ-P,et al. Directcomparisonof a

radioiodinatedintact chimericanti-CEAMab with its F(ab')2 fragmentinnude mice bearing different colon cancer xenografts. BrJ Cancer 1993;68:684—690.

32. ButheggerF, PIlegrinA, HardmanN, et al. Differentbehaviourof mousehumanchimericantibodyF(ab')2fragmentsof IgGi, 1g02andIgG4subclass in vivo. Intl Cancer 199250:416-422.

33. HammarstromS, Shive@yJE, PaxtonRi, et aLAntigenicsites in carcinoembryonicantigen.CancerRes1989;49:4852-4858.

34. Nap M, HammarstrOmML, BOrmer0, et al. Specificityand affinityofmonoclonalantibodiesagainstcarcinoembtyonicantigen.CancerRes 1992;52:2329—2339.

35. BucheggerF, SchreyerM, CarrelS, Machi-P. Monoclonalantthodiesidentify a CPA crossreacting antigen of 95 kD (NCA.95) distinct in antigenicityand tissuedistributionfromthe previOUslydescribedNCAof55 kD.Intl Cancer198433:M3-649.

36. Roth IM, Brostoff 3, Male DK. Mtil,ody structure and function. In:&nnett D, ed. Immunology.London,England:GowerMedicalPublishingLtd; 1985:5.1—5.10.

37. FritscheR, MachJP. Isolationandcharacterizationof carcinoembiyonicantigen (CEA) extracted from normal human colon mucosa. Immunochem1977;14:119—127.

38. Buchegger F, Mach i-P. Different SOlid-phaseenzyme immunoassays usingfive monoclonal antthodies reactingwith distinct epitopes of carcinoembryorik antigen. In: Avrameas S, et aL, ads. Immuncenzymatic Techniques.Amsterdam,TheNetherlands:ElsevierSciencepublishersB.V.;1983:385—394.

39. DeNardOGL, DeNardO Si, Miyao NP, Ctal. Non-dehalogenation mochanismsforexcretionofradioiodineafteradministrationoflabeledantibodies.JntlBiolMai*ers 1988;3:i-9.

40.JamRK,BaxterLI. Mechanismsofheterogeneousdistributionofmono

428 TheJournalofNudearMediane@ Vol.36@ No.3@ March1995

donal antibodies and other macromolecules in tumors: significance of dcvated interstitialpressure. CancerRes 1988;48:7022—7032.

41. Folli S, PIlegrin A, Chalandon Y, et al. lumor necrosis factor can enhanceradio-antibody uptake in human colon carcinoma xenografts by selectiveincreaseofvascular permeability.Intl Cancer 199353:829-836.

42. FujimonK, CovellDO,FletcheriE,WeinsteinJN.A modelinganalysisofmonoclonalantibodypercolationthroughtumors: a binding-sitebarrier.INuci Med 1990;31:1i91—1198.

43. HaskellCM,BucheggerF, SchreyerM, CarrelS, Machi-P. Monoclonalantibodies to carcinoembryonic antigen: ionic strength as a factor in theselection of antibodies for iminUnOScintigraphy. CancerRes 1983;43:3857-3864.

44. HunzikerW, FumeyC. A di-leucinemotifmediatesendocytosisand baso.lateral sorting of macrophage IgG Fc receptors in MDCK cells. EMBO I1994;13:2963—2969.

45. Folli S, WagniIres 0, PIlegrin A, Ct al. Localization and detection offluoresceinatedchimeric antibodies against carcinoembiyonicantigen inprimarycolorectalcarcinomas:firstapproachto clinicalimmunophotodiagnosiLProc NatlAcad Sd USA i992;89 973-7977.

46. HaganPL, HalpernSE, DillmanRO, et al. lumor size: effecton monoclonal antibody uptake in tumor models. INuciMed 1986;27:422-427.

47. AkabaniG, PostonSrJW,BolchWE.Estimatesofbeta absorbedfractionsin small tissuevolumes for selected radionuclides.JNuclMed 199l;32:835-839.

48. HumznJL Dosimetricaspectsofradloinbeledantibodiesfortumortherapy.INuciMed 1986;27:i490-1497.

49. Fowler JF. Radiobiological aspects oflow dose rates in radioimmunotheral,),.InJRadia@ OncolBk,!Phys 1990;18:1261-1269.

429ChimencversusMouseMAbin Patients•Bucheggerat Si.