Complementation of intracavitary and intravenous administration of a monoclonal antibody (B72. 3) in...

ICANCER RESEARCH 47, 4218-4224, August I. 1987]

Complementation of Intracavitary and Intravenous Administration of a Monoclonal

Antibody (B72.3) in Patients with CarcinomaDavid Colcher, Jose Esteban, Jorge A. Carrasquillo, Paul Sugarbaker,1 James C. Reynolds, Gail Bryant,Steven M. Larson, and Jeffrey Schlom2

Laboratoiy Tumor Immunology and Biology [D. C., J. E., J. S.J, and Surgery Branch [P. S.J, and Laboratory of Pathology /G. BJ, National Cancer Institute, andDepartment of Nuclear Medicine {J. A. C., J. C. R., S. M. L.], Clinical Center, NIH, Bethesda, Maryland

ABSTRACT

Monoclonal antibody (MAb) B72.3 has been shown to have selectivereactivity for a wide range of carcinomas (colorectal, ovarian, breast,lung, gastric, and endometrial) versus normal adult tissues. ' "I-l .abelcd

B72.3 IgG has recently been shown to selectively bind carcinoma lesionswhen administered i.v. in patients with metastatic colorectal cancer. Wereport here the first direct comparison of i.p. administered |' "IJB72.3

IgG to specifically localize metastatic carcinoma. Three of 10 patientsstudied were negative for tumor detection by both CAT scan and X-raybut were positive for tumor localization via y scanning of i.p. administered"'I-labeled MAb B72J IgG. Direct analyses of biopsy specimens of

carcinoma and normal tissues demonstrated ratios of greater than 70:1(based on percentage of injected dose/mg) for tumor MAb localizationversus normal tissues. Specificity of [I3II|B72.3 tumor targeting was

demonstrated by the concomitant administration of an equal dose of an'"I-labeled isotype identical (IgGi) control MAb. Simultaneous i.p.administration of (I3II]B72.3, and i.v. administration of |''M|B72.3 in

individual patients demonstrated: (a) peritoneal implants are targetedmore efficiently via i.p. MAb administration, and (b) hematogenouslyspread and lymph node mÃ©tastasesas well as local recurrences aretargeted more efficiently by i.v. administered MAb. No antibody toxicitywas observed in any patients. Pharmacokinetics of MAb clearance demonstrated that only 10 to 30% of the i.p. administered MAb was foundin plasma. These studies thus demonstrate the efficacy of intracavitaryMAb administration as well as the advantage of the concomitant use ofintracavitary and i.v. administered MAbs for tumor targeting and forpotential MAb guided therapy of metastatic carcinoma.

INTRODUCTION

MAb3 B72.3, developed using a carcinoma biopsy extract as

immunogen, has previously been shown via radioimmunoassayand immunohistochemical analyses to be reactive with themajority of biopsy specimens of primary and metastatic colorectal, ovarian, breast, lung, gastric, and endometrial carcinomas (1-5). The reactive high molecular weight glycoproteinantigen (termed TAG-72) has recently been purified and characterized (6). MAb B72.3 has been extensively studied forreactivity to a wide range of normal human tissues. The onlytissues expressing TAG-72 in amounts similar to those ofcarcinoma are fetal gut and the lumen of some ducts of secretoryendometrium; proliferate and resting endometrium, however,were negative for TAG-72 expression (7, 8). The extremeselective reactivity of MAb B72.3 for carcinoma versus normaladult tissues (including normal and reactive mesothelium) hasled to its use to detect carcinoma cells in pleura! effusions andfine needle aspiration biopsies (9-12).

Received 4/10/87; accepted 5/4/87.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.

1Present address: Director of Surgical Oncology Emory Clinic, Atlanta. GA

30322.2To whom requests for reprints should be addressed, at National Cancer

Institute, Laboratory' of Tumor Immunology and Biology, Building 10. Room8B07, Bethesda, MD 20892.

3The abbreviations used are: MAb, monoclonal antibody; TAG, tumor asso

ciated glycoprotein: RIA, radioimmunoassay; % ID/mg, percentage of injecteddose per milligram; Rl. radiolocalization index.

A clinical trial has recently been completed in which 27patients with suspected or confirmed colorectal cancer wereadministered "'I-labeled B72.3 IgG i.v. These studies demonstrated the ability of [I31I]B72.3 IgG to selectively localize

primary and metastatic colorectal lesions via direct analyses ofbiopsy specimens. Ratios of MAb uptake for tumor versus awide range of normal tissues (on a per milligram basis) rangedfrom 3:1 to 40:1 for 70% of carcinoma lesions examined (13,14). Recent trials have also demonstrated similar results when[U'I]B72.3 IgG was administered i.v. to localize ovarian cancerlesions.4

In the study reported here, we set out to: (a) determine thefeasibility of i.p. administration of radiolabeled B72.3 for tumorlocalization (via both gamma scanning and direct analyses ofbiopsy specimens); (b) determine the specificity of tumor localization by the concomitant i.p. administration of [UII]B72.3and an isotype matched control [l25I]MAb BL-3 (anti-idiotype)

IgG; (c) compare tumor localization of i.v. versus i.p. administered MAb by the simultaneous administration of [125I]B72.3IgG i.v. and ['"I]B72.3 IgG Â¡.p.;and (</) define the pharmaco-

kinetics of plasma clearance of both i.p. and i.v. administeredradiolabeled MAb B72.3.

MATERIALS AND METHODS

Patients. Ten patients with colorectal carcinoma were selected fromongoing NCI-Surgery Branch protocols that examined the efficacy ofchemotherapy after surgical removal of the bulk of i.p. tumor. Nomodifications of this protocol were made for this study. The patientshad metastatic colorectal carcinoma whose primary lesion had beenpreviously resected. Some patients had received additional forms oftherapy. All patients were included in the protocol for staging and/ordebulking of metastatic disease. The patients age ranged from 16 to 63years (seven males, three females). Histologically, the tumors includedpoorly differentiated adenocarcinomas, mucinous adenocarcinomas,and signet ring cell variants.

The patients received '"I-labeled B72.3 IgG i.p. and an additional

MAb i.v. or i.p. as detailed below. Approximately 6 to 8 days postin-jection of the radiolabeled antibodies, the patients underwent surgicalexploration and resection of the tumor with normal tissues resected forstaging purposes.

Monoclonal Antibodies. MAb B72.3 is a murine IgGi obtained byimmunization of mice with a membrane-enriched fraction of a humancarcinoma metastasis to the liver. Its generation, characterization, andreactivities have been described in detail elsewhere (1-3). For this study,B72.3 IgG was purified from ascitic fluid by ammonium sulphateprecipitation and ion-exchange chromatography as previously published (15). The purified IgG was filtered and all end lots were testedfor the lack of mycoplasma, 12 adventitious viruses, and lack of pyro-genicity, as well as for sterility and general safety.

In selected patients, an isotype matched control MAb (BL-3) wasused. BL-3 is an antiidiotype MAb that reacts with a human H celllymphoma. Prior to its use in our studies, it was tested in RIA forreactivity with extracts of eight colon carcinomas and 31 normal adulttissue extracts including normal human colon, liver, spleen, red bloodcells, polymorphonuclear leukocytes, and kidney, etc., and was shown

4 Personal communications.

4218

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COMPLEMENTATION OF i.p. AND i.v. ADMINISTERED MAbs

to be non-reactive. BL-3 IgG was tested for sterility, etc., as describedabove, before use in the clinical trial.

lodination of MAbs. B72.3 was labeled with Na'" I using the lodogen

method (16). Purified IgG (1500 pg) was labeled with approximately15 mCi of Nal31I in a glass vial coated with 250 Â¿igof lodogen. After a10-min incubation, the free '"I was removed by gel filtration through

a Sephadex G-25 column. The labeled MAb was then diluted in salinecontaining 1% human serum albumin. Labeling efficiency was approximately 60%, and the specific activity ranged from approximately 6-11mCi/mg of antibody. B72.3 IgG and BL-3 IgG were similarly labeledusing Na'25I.

The immunoreactivity of the radiolabeled antibody was tested in asolid phase RIA with colon tumor extracts (LS-174T) known to reactwith B72.3 (15, 17). Prior to administration to patients, unlabeledB72.3 IgG was added to the radiolabeled MAb to obtain the desiredspecific activity; the sample was then packaged into sterile ampules,shown to be apyrogenic using the Limulus amebocyte lysate assay, andthen tested for sterility.

Antibody Administration and Sample Collection. The patients receivedfrom 0.76 to 1.2 mg of I31l-labeled MAb with activities ranging from 5

to 10 mCi (see Table 1) by a bolus infusion into the peritoneal cavitywith approximately 1 liter of 1.5% Inpersol (Abbott Laboratories, N.Chicago, IL) via a Tenckhoff catheter. In selected patients an isotypecontrol, MAb, BL-3, was coinjected i.p. (approximately 1.0 mg and 2mCi); or '"I-Iabeled B72.3 IgG was injected i.v. (approximately 1.0 mg

and 2 mCi) to examine the specificity of the i.p. route of administration.Patient Imaging. The patients were imaged within 2 h of MAb

administration and daily up to surgery. A 7 camera, with a large fieldof view and a high energy collimator, was utilized. A 20% window overthe 364 keV 7-ray of 131Iwas used to obtain anterior and posterior

whole body images as well as multiple spot views (5 to 10 min each).In addition to analogue images, digital images were recorded with aHewlett-Packard Scintigraphic computer.

Tissue Studies. All the tissues removed at surgery were labeledaccording to organ and anatomic location and a representative specimenof each was immediately weighed in an analytical balance and countedin a 7 counter in order to establish the activity (cpm) per gram of tissue.Those specimens were then fixed in formalin, embedded in paraffin,and sectioned for routine histolÃ³gica!studies. All sections were studiedby light microscopy and the percentage of tumor present in each sectiondetermined. Representative nontumor tissues were selected and theiractivity in counts per gram averaged. The uptake of the radionuclide(% ID/mg, i.e. % ID/g x IO3) in historically confirmed tumor

(containing over 20% tumor cells) was divided by the value obtainedfor histologically normal tissues providing the RI for each tissue andeach radionuclide. Metastatic lesions were examined independently bythree pathologists and characterized as (a) peritoneal implants or as(b) nonimplant mÃ©tastases,i.e., hematogenously borne mÃ©tastases,lymph node mÃ©tastases,or local recurrences. For those lesions whereall three pathologists did not arrive at the identical conclusion thelesion was listed as unclassified (see Table 8).

RESULTS

Study Parameters and -y Scanning. As seen in Table 1, ages

of patients ranged from 16 to 63 years. All patients in this studywere part of a preexisting National Cancer Institute Surgery

Branch protocol for the surgical resection of metastatic colo-rectal cancer lesions followed by i.p. administration of therapeutic agents. At the time of entry into the protocol, all patientshad confirmed or suspected metastatic peritoneal lesions. Fourto 7 days prior to surgery, all patients were administered I3II-labeled B72.3 IgG i.p. via a catheter (see "Materials and Methods").

Previous studies using i.v. administered I31l-radiolabeled

B72.3 IgG have shown that differences in administration ofbetween 0.2 and 20 mg of IgG or between 0.8 and 10 mCi ofI31I(with a specific activity range of 0.3 to 11.8 mCi/mg) had

little or no effect on the radiolocalization of colon carcinomalesions as determined by 7 scanning or direct analysis of tumorsamples (% ID of MAb bound per gram of tissue) (13, 14).Therefore, as seen in Table 1, all patients received between 0.76and 1.2 mg of IgG and between 5.0 and 10.0 mCi 13II, withspecific activities of [131I]B72.3 IgG ranging from 6.6 to 11.0

mCi/mg.On the day of, or prior to surgery, patients were scanned with

a 7 camera; all scan results were interpreted prior to surgery.Scans (7) of seven of 10 patients showed clearly discernibleconcentrations of radiolabeled MAb in various distinct regionsof the peritoneum (Table 1). In all such cases, these lesionswere identified at surgery as tumor and later confirmed ascarcinoma via histopathological examination (Table 1). A representative positive 7 scan is shown in Fig. 1, A-C (patientMM). Note the accumulation of radiolabeled MAb in the"gutters" of the peritoneum immediately after i.p. MAb admin

istration (Fig. \A) and at 3 days post-MAb administration (Fig.IB). The 7 scan at 6 days post-[13'I]B72.3 administration,

however, clearly shows MAb concentration in distinct areas ofthe peritoneum (confirmed as cancer via surgery and histopa-thology) (Fig. 1C). These findings can be contrasted with thenegative 7 scans of patient RK (Fig. 1, D-F). As will be furtherdetailed below, patient RK was a patient without any i.p.disease.

Three of the 10 patients studied (NJ, WJ, and JB) werepositive for MAb localization via 7 scanning (areas confirmedas tumor at surgery), but were negative for tumor via computerized axial tomography scanning and X-ray studies. Lesionsas small as approximately 1.5 cm in diameter were clearlydefined via 7 scans.

Direct Analyses of Biopsy Specimens. All specimens removedat surgery were immediately coded, weighed, placed in a 7counter, and analyzed for cpm/mg of tissue. Specimens werethen fixed, embedded, and analyzed for percentage of carcinomacells present. Table 2 shows representative data from one patient (patient MM, whose scan is seen in Fig. 1, A-C); histologically confirmed normal tissues, removed for staging purposes, show between 0.11 and 0.36% injected dose (%ID) of[13II]B72.3 MAb per mg (i.e., % ID/g x IO3) of tissue. By

contrast, the various carcinoma lesions of different sites showed

Table 1 Gamma scan results of patients administered [>3>I]B72.3 IgG i.p.

NameMMMHRKNJWJSMJBEPMGJKAge36165433516336636038SexMFMFMMMMFMmg0.760.810.810.870.890.940.890.951.001.10mCi5.08.98.97.68.39.910.010.06.89.9Specificactivity6.611.011.08.79.310.511.210.56.89.0ScanresultsPathology+

MucinousSignet-

Mucinous+Mucinous+Mucinous+Mucinous+Mucinous+

MucinousPoordif.+

MucinousLocationDiffuse

peritoneum, appendix,cecumOvary,intestinalserosaCecum,ileum, lymph node, intestinalserosaDiffuse

peritoneumDiffuseperitoneumDiffuseperitoneumDiffuse

peritoneum,rectosigmoidDiffuseperitoneumColon,

ileum, lymphnodeDiffuseperitoneum

4219




RADIOIMMUNOSCINTIGRAPHY OF INTRAPERITONEALLY ADMINISTERED131I-B72.3 IgG IN PATIENTS WITH COLORECTAL CANCER

Table 3 RI of patients injected i.p. with f'3'IfB72.3

IMMEDIATE

PATIENT RK

IMMEDIATE 3 DAYS 7 UAYb

Fig. 1. Radioimmunoscintigraphy in patients with metastatic colorectal cancerafter i.p. administration of |'"I)B72.3 IgG. Two patients were injected i.p. withradiolabeled B72.3 IgG as described in "Materials and Methods." Patients RK

and MM were scanned immediately after antibody administration and variouslimes.

Table 2 Administration (i.p.) of[nil]B72.3 IgG (patient MM)

TissuedescriptionCarcinomasPeritoneal

implantsIntraabdominaltumor ( V =4)Lesser

sacGastrohepaticSplenic

flexure (/V=2)SmallbowelserosaPeriaorticDiaphragm

undersurfaceTailpancreasSerosacolonGastricarteryOmentumPancreas

surfaceIleumlymph node (N =2)Transferse

colon serosa (yV=2)FalciformligamentSigmoidserosaLiver

domeNonimplantsCecum

& granulationtissueSpleniccapsuleAppendixIleum

and adiposetissueNormalAdipose

tissue (N =2)ColonIleumLymph

node (N =5)Skin&fatSofttissue fÃstulatract%

ID/mg16.7513.7213.1611.479.847.146.816.076.065.885.034.492.081.661.521.420.940.850.600.480.41O.I

I0.150.260.220.240.36RI"66.554.552.345.639.128.427.124.124.123.420.017.88.36.66.05.63.73.42.41.91.60.40.61.00.90.91.4Tumor

(%)909095908520858090708080909025857060804070

" RI was determined by dividing the % ID/mg of the tumors by the averaged

% ID/mg of normal ileum and lymph nodes.

between 0.41 and 16.75% ID/mg. These data can also beexpressed as RIs, i.e., the ratio of the % ID/mg in tumor versusthe % ID/mg of normal tissues. As seen in Table 2, the RIs ofthe histologically confirmed normal tissues range from 0.4 to1.4 while those of the carcinoma lesions range from 1.6 to 66.5,with most tumors (23 of 27) having RIs of greater than 3.

Table 3 shows the RIs (based on % ID MAb uptake per mgof tissue) of the biopsy specimens of tumor and normal tissuesfrom the 10 patients receiving i.p. administered MAb B72.3.An RI of >3 was arbitrarily chosen as a "positive" radiolabeled

uptake. Eighty-three of 112 (74%) of carcinoma lesions showed

TumorNameNJMHRKMMMGWJEPSMJBJKTotal

no. oflesions%RI:<30254112050029263-1014350776003329>101700180321635045<31210811725161239599Normal3-10000000001011>10000000000000

RIs of >3. In some patients with a large tumor burden, as muchas 40% of the injected dose was found bound to carcinoma.Note that of the 29 lesions negative for MAb uptake, 11 werefrom patient MG, the only patient in which all tumor lesionswere negative for TAG-72 antigen expression. Evidence will bepresented below (and in Table 8), as to why at least 10 of the18 remaining carcinoma lesions did not appreciably take up theÂ¡.p.administered B72.3 MAb. Of the 95 histologically confirmed normal tissues biopsied, all but one demonstrated RIsof <3 (Table 3). The one biopsy not in this category had an RIof 3.5 and was a histologically normal spleen from patient JB.It is interesting to note that this patient had the highest levelof circulating antigenâ€”['"IJB72.3 IgG immune complex as

determined by high performance liquid chromatography andRIA (18). The other 94 normal tissue biopsies with RIs of <3were from numerous sites including colon, spleen, ileum, pancreas, lymph node, fallopian tube, liver, ovary, duodenum, andkidney, with the vast majority of RIs ranging from 0.5 to 1.5.

Specificity of MAb B72.3 Radiolocalization. To determine ifthe radiolocalization observed with the i.p. administered [I3II]-

B72.3 IgG was specific, four patients received concomitantinfusions (administered i.p.) of [mI]B72.3 IgG and '"I-labeledcontrol MAb BL-3 IgG. B72.3 and BL-3 were administered atthe same milligram dose and time for each patient. Table 4(patient WJ) shows representative results obtained. As can beseen, the % ID/mg of the [13II]B72.3 ranged from 0.27 to 11.0

for the carcinoma biopsies, with RIs ranging from 0.9 to 37.4times those seen in normal tissues, with most (10 of 12) havingRIs greater than 3. The % ID/mg observed for the ['"I]BL-3,

on the other hand, ranged from 0 to 0.46 for the same carcinomalesions with RIs of only 0 to 1.3. The ratios of % ID/mg forB72.3 to BL-3 for the 10 carcinoma lesions examined werefrom 5.5:1 to >100:1 for tumors showing MAb localization(Table 4). Similar results were obtained for the other threepatients studied. There were, however, some important observations obtained in these analyses concerning "nonspecific"

MAb uptake. As shown in Table 5, MAb BL-3 uptake wasnegative (i.e., RI < 3) in 28 carcinoma lesions, but showed RIsof >3 in 11 others. When one analyzes the ratios of % ID/mgof these lesions for B72.3 versus BL-3 (Table 5) one observesthat in none of 39 lesions was BL-3 uptake greater than that ofB72.3; in eight lesions they were comparable and in 31 of 39lesions the B72.3/BL-3 ratios were greater than 2:1, with ratiosof greater than 10:1 in the majority of cases; ratios of > 100:1,moreover, were observed for many lesions. Thus, while theseresults demonstrate the specificity of the B72.3 binding, theydo point out that "nonspecific'" binding of irrelevant MAb to

carcinoma lesions may and does indeed occur at times.Simultaneous i.p. and i.v. Administration of MAb B72.3. Stud

ies were next conducted to determine the relative efficacies of4220




Table 4 Concomitant i.p. administration of[n'IJB72.3 IgG and {'"IJBL-3 control IgG (patient WJ)

TissuedescriptionCarcinomaPeritoneal

implantsJejunummesenterySigmoidcolonRighthemidiaphragmTransverse

colonmesenterySplenichilum (N =2)Splenic

flexureSofttissue nodules (N =2)JejunumIleac

artery (N =2)NormalSpleen

(N =2)Mesentery,implant, smallbowelImplant,

jejunum (n =5)IleocolicanastomosesImplant,ileumPelvis,perivesicalSpermaticarteryAdhesions,

small bowel (N =2)Softtissue (N =7)Omentum

(N =2)PancreasSplenic

hilumRI"131I(%

ID/mg) 125I(% ID/mg) I3IIU5I10.988.265.932.472.391.851.260.970.270.440.360.300.280.260.230.230.210.210.110.100.09"

RI was determined by dividing the % ID/mg of the tumors by the averaged %0.38

37.41.10.0028.10.00.4320.21.20.32

8.40.90.468.21.30.256.30.70.134.30.40.123.30.30.200.90.60.13

1.50.40.441.21.20.261.00.70.331.00.90.270.90.80.300.80.90.300.80.80.220.70.60.260.70.70.090.40.20.120.30.30.110.3 0.3Ratio(%

ÃŒD/mg)28.6>

100.013.67.85.57.39.48.01.33.30.81.60.81.00.80.80.90.81.50.80.8rumor(%)603090709595583075ID/mg

of the normal peritonealimplants.Table

5 Patients injected i.p. concomitantly with (131I}B72.3and['"IJBL-3No

ofNametumorsB72.3WJ

122EP90SM

125JB60Total

397%B72.318%

BL-3"Ratios

of % ID/mg.Radiolocalization

index<3

3-10BL-3

B72.3BL-312

7027612

6020028

2065172

15Table

6 Concomitant administration of[131{JB72.3i.p.Tissue

descriptionCarcinomaOmentumMesentery,

sigmoidcolonPeritonealliver capsule (N =4)PeritonealgastroesophogealjunctionOmentum,

greaterPeritonealperipancreasOmentum,

lesser (N =2)Diaphragm(N =3)Peritoneal

spleen, capsule (N =4)NormalAdipose

tissueMesentery,transversecolonPeritoneum,

rightPeritonealadhesions (JV=3)Abdominal

scarFalciformligamentSpleen

(N = 3)>10B72.3

BL-3<0.53002

1010064012

5031013Ratios

ofB72.3/BL-3"0.5-2

>2>42005

3110200183421

810>10100952462and

{'"IJB72.3 i.v. (patient NJ): advantage of i.p.route%

ID/maratios131I(i.p., % ID/mg) '"I (i.V., % ID/mg)("'I/"5!)44.5140.0439.6239.2711.4145.2727.0827.2329.300.070.520.841.210.991.042.705.85

7.615.427.399.195.648.454.652.75

4.1611.304.017.543.687.76

3.4810.722.770.05

1.310.491.070.661.260.781.560.472.090.372.823.45

0.78RI"i.p.

131I i.v.1MI32.6

12.229.311.329.019.128.8

17.68.45.733.223.519.815.719.916.221.5

22.30.1

0.10.41.00.61.40.91.60.71.00.80.82.0

7.2Tumor8080668040856368690000000' Determined by dividing the % ID/mg of the tumors by the averaged % ID/mg of all normal biopsies.

i.p. versus i.v. administered MAb to localize tumor lesions. Toachieve this goal, patients were concomitantly administered13ll-labeled B72.3 i.p. and '"Â¡-labeled B72.3 i.v. Both the i.p.

and i.v. administered MAb preparations in each of four patientswere identical as far as mg dose, and radiolabeling conditions.In 35 of 55 carcinoma lesion biopsies obtained, the i.p. administered B72.3 localized at least two times better in terms of %ID/mg than the i.v. administered MAb. In seven lesions, MAb

localization was comparable via either route, and in 13 lesions,the i.v. administered MAb B72.3 localized at least two timesbetter than the i.p. administered MAb. Representative resultsfor each situation are shown in Tables 6 and 7. As seen forpatient NJ (Table 6), % ID/mg taken up by carcinoma lesionsranged from 11.4 to 45.3 for i.p. administered B72.3 (I31I)

versus values of only 2.8 to 11.3 % ID/mg for the i.v. administered B72.3 (125I).The ratios of uptake of the i.p. administered

4221




Table 7 Concomitant administration of['"IJB72.3 i.p. and f'"l}B72.3 i.v.

(patient RK): advantage of i.v. route

Ratios, RI"

'"I (i.p., I2*I(i.V., mg i.p. i.v. TumorTissue description % ID/mg) % ID/mg) (I3'I/'"I) '"I '"I (%)

CarcinomaOmentumPeritoneal

implant.serosaRetroperitoneumCecum

(N =4)Lymphnode.ileocecumNormalColonlleumLymph

nodes(<V=3)Omentum

(N =2)Transversecolonserosa0.300.210.350.260.060.110.110.050.100.071.422.332.862.860.330.120.150.180.040.200.20.10.10.10.20.90.70.32.80.42.43.34.02.90.61.31.30.61.10.87.612.615.415.41.80.70.81.00.21.14040705340

" RI was determined by dividing the % ID/mg of the tumors by the averaged

% ID/mg of normal ileum and lymph nodes.

MAb were from 2.8 to 7.6 times greater for individual lesionsthan the i.v. administered MAb (Table 6). Conversely, as seenin patient RK (Table 7), % ID/mg values were from five to 11times greater for the i.v. administered MAb than that of the i.p.administered MAb. Note the levels of uptake in the normaltissues for both patients NJ and RK (Tables 6 and 7) weresimilar.

In an attempt to define the reason(s) for the divergence inuptake of i.p. and i.v. administered MAb among differentcarcinoma lesions, three pathologists independently examinedthese lesions and characterized them as to various properties(two of the pathologists were not told the reason for thisexercise). The most striking correlation with differential MAbuptake was whether a given metastasis was a peritoneal implantor "nonimplant." Metastatic lesions were characterized as: (a)

peritoneal implants, or (b) nonimplant mÃ©tastases,i.e., hema-togenously borne mÃ©tastases,lymph node mÃ©tastasesor localrecurrences. For those lesions where all three pathologists didnot arrive at the identical conclusion the lesion was listed as"unclassified." As seen in Table 8, in all 10 nonimplant mÃ©tas

tases from three patients, the ratios (% ID MAb/mg) of uptakeof the i.v. injected B72.3 was at least two times that of the i.p.administered MAb, with ratios ranging up to 38:1. Conversely,in 35 of 40 lesions classified as peritoneal implants, i.p. administered B72.3 localized at least two times better than i.v. administered MAb with the ratios obtained for the majority of lesionsbeing greater than 5:1 (Tables 6 and 8). Five of 40 peritonealimplants bound comparable levels of i.p. and i.v. administeredB72.3.

Pharmacokinetics of Plasma Levels following i.p. MAbAdministration. Plasma samples from eight patients studied

were obtained prior to MAb administration and at various timepoints post MAb administration. The plasma clearance of thei.v. administered [125I]B72.3 IgG and the [125I]BL-3control IgG

were similar, with approximately 50% of the injected dose inthe plasma at day 2 and approximately 10% of the injecteddose in plasma at day 7 post-MAb administration (Fig. 2). Incontrast, no more than 30% of the injected dose of i.p. administered B72.3 IgG or BL-3 IgG appeared in the plasma at anypoint in time, with peak values being obtained at days 2 to 3(Fig. 2).

Pre-MAb administration plasma samples were tested for theB72.3 reactive antigen TAG-72 via RIA as previously described(18, 19). The appearance of ["'I]B72.3 IgG in the plasma was

noted to be reduced in four of eight patients studied (Fig. 2,closed circles), MH, RK, SM, EP, in that ['"I]MAb levels inplasma did not rise above 10-12% of injected dose. Analysis ofvarious parameters revealed that sera from these four patientsshowed the highest levels of circulating TAG-72 antigen [valuesof 95, 15, 40, and 78 units/ml (18, 19), respectively]. Theimplications of this observation for the MAb guided therapywill be discussed below.

DISCUSSION

MAb B72.3, utilized in the studies reported here, has previously been shown to have a high degree of selective reactivityto a wide range of carcinoma lesions including colon and rectalcarcinoma, mucinous and ovarian carcinoma, mammary carcinoma, adeno- and adenosquamous lung carcinoma, gastric carcinoma, and endometrial carcinoma (1-5). Preclinical studiesusing radiolabeled B72.3 to target human tumor xenografts inathymic mice have demonstrated that this MAb remains boundto tumor for up to 19 days (15-16). Previous i.v. administrationof ['"I]B72.3 IgG to 27 patients have demonstrated selective

in vivo tumor targeting and lack of any toxicity (13, 14). Severali.v. administered MAbs have now been used in clinical trials totarget carcinoma and melanoma lesions (20-29). A recent studyreported the use of an MAb to human milk fat globule(HMFG2) to target ovarian carcinoma via intracavitary administration (30, 31). This study, however, (a) did not confirmtumor localization by direct analysis of tumor tissue (or viaanalysis of % ID/mg bound to tumor); (b) did not employ acontrol MAb to demonstrate MAb binding specificity; (c) didnot compare i.v. and intracavitary administration of MAb; and(d) did not monitor pharmacokinetics of MAb plasma clearance.

In the studies reported here we demonstrate the advantage ofMAb B72.3 to target peritoneal implant carcinoma mÃ©tastaseswhen administered i.p., and the converse advantage of B72.3 to

Table 8 Ratio of% ID/g of i.p. versus i.v. administered B72.3 IgGMetastalic lesions showing specific antibody localization were examined independently by three pathologists and characterized as (a) peritoneal implants or as (b)

nonimplant mÃ©tastases,i.e., hematogenously borne mÃ©tastases,lymph node mÃ©tastases,or local recurrences. For those lesions where all three pathologists did notarrive at the identical conclusion the lesion was listed as unclassified.

Pathology ofmÃ©tastasesNonimplant

Hem/LN/Rec"NameMMNJ

MHRKTotal%>20

000000.5-20

00000<0.52

035

10100>219

160

03588Implant0.5-23

2005

12<0.50

00000>20

00000Unclassified0.5-20

0202

40<0.50

0123

60' Hem. hematogenous mÃ©tastases:LN. lymph node mÃ©tastases;Ree. recurrence.

4222




PLASMA CLEARANCE OF 872.3 IgGPatients Injected Both IP and IV

IP w/ Ag

REFERENCES

Fig. 2. Plasma clearance of B72.3 IgG after administration both i.v. and i.p.in patients with colorectal cancer. Patients were injected i.v. with [125I]B72.3IgGand i.p. with [13II]B72.3 IgG. Plasma samples were drawn at various times. â€¢and*, MAb plasma clearance levels for patients receiving i.p. administered MAb. â€¢,average of four patients with low levels of TAG-72 serum antigen; *, average offour patients with high levels of TAG-72 serum antigen. The average clearanceof i.v. administered MA6 for both classes of patients is given for comparison (â€¢).

target hematogenously spread and regional lymph node mÃ©tastases as well as local recurrences when administered i.v. Thespecificity of the B72.3 binding was demonstrated in the dualMAb administration experiments in which B72.3 was shown tobind from four to over 100 times more efficiently to carcinomalesions than control MAb. These same studies did demonstratethe potential of a nonspecific MAb (in this case BL-3) to betaken up by carcinoma lesions when administered i.p. Thesefindings should be kept in mind in the evaluation of previousand future clinical trials.

There are several diagnostic implications from the studiesreported here. Radiolabeled B72.3 may now be considered foruse in the localization of suspected colorectal or ovarian (4)carcinoma lesions. Radiolabeled B72.3 can also be used, alongwith the serum assay (18, 19) for the presence of TAG-72antigen, to monitor efficacy of various therapies. The studiesreported here demonstrated that carcinoma was detected by[I3II]B72.3 but could not be detected by computerized axial

tomography scan or other radiographie procedures in three ofthe 10 patients studied; lesions as small as 1.5 cm were detected.Furthermore, efficiency of detection should improve whenradionuclides such as "Te and '"In, with more favorableimaging characteristics, are substituted for 13II.

These studies have also demonstrated the potential of i.p.administered B72.3 as a therapeutic agent, either coupled todrugs, effector cells, or radionuclides. Direct analyses of biopsyand plasma specimens have permitted dosimetry calculations(32) that indicate that if higher doses of [I31I]B72.3 IgG are

employed, sufficient radiolabeled MAb may be delivered totumor masses for cell killing with minimal toxicity to normaltissues. Efficiency of killing will increase, moreover, when moreefficient cytotoxic radionuclides such as *Â°Yare coupled to

B72.3; studies to develop such reagents are currently in progress. The studies reported here have also shown that only aminor component of the i.p. administered radiolabeled MAbIgG is found in the plasma (Fig. 2), an important point toconsider if one wishes to minimize plasma borne radiolabeledMAb as a potential source of marrow toxicity. Thus, thesestudies have demonstrated the necessity of considering eithersole use of intracavitary administered MAb or the concomitantuse of Â¡ntracavitary and i.v. administered MAb in protocolsaimed at MAb-guided diagnosis or therapy of a wide range ofhuman carcinomas.

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Metastatic colon carcinoma detected with radiolabeled F(ab')2 monoclonal antibody fragments.Radiology, 149: 549-555. 1983.Mach. J-P., Chatal, J-F., Lumbroso. J-D., Buchegger, F.. Forni, M., Ritschard, J., Berche, C., Douillard. J-Y., Carrel. S., Herlyn, M., Steplewski,Z., and Koprowski, H. Tumor localization in patients by radiolabeled monoclonal antibodies against colon carcinoma. Cancer Res., 43: 5593-5600,1983.Hnatowich, D. J., Griffin, T. W., Kosciuczyk, C., Rusckowski, M., Childs,R. L., Mattis, J. A., Shealy, D., and Doherty, P. W. Pharmacokinetics of anIndium-111 labeled monoclonal antibody in cancer patients. J. NucÃ.Med.,26:849-858. 1985.Chatal, J-F., Saccavini, J-C. Fumoleau, P., Doulliard, J-Y., Curtet, C.,Kremer, M., LeMevel, B., and Koprowski, H. Immunoscintigraphy of coloncarcinoma. J. NucÃ.Med., 25: 307-314. 1984.

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4224



(CANCER RESEARCH 47, 6161, November 15, 1987]

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Erratum

The article "Complementation of Intracavitary and Intravenous

Administration of a Monoclonal Antibody (B72.3) in Patients withCarcinoma," by David Colcher et al., which appears on pp. 4218-4224

of the August 1, 1987, issue, contains errors in Tables 2, 4, 6, and 7and the references to these tables in the text. The units are specified in

"Materials and Methods" as % ID/mg and are defined as % ID/g xIO3; % ID/g x IO3 is actually % ID/kg. This section of the text, the

listings in Tables 2, 4, 6, and 7, and the references to those tables inthe text should be adjusted accordingly.

6161

1987;47:4218-4224. Cancer Res David Colcher, Jose Esteban, Jorge A. Carrasquillo, et al. with CarcinomaAdministration of a Monoclonal Antibody (B72.3) in Patients Complementation of Intracavitary and Intravenous

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