[CANCER RESEARCH 45, 5769-5780, November 1985]

Definition by Monoclonal Antibodies of a Repertoire of Epitopes on

Carcinoembryonic Antigen Differentially Expressed in Human

Colon Carcinomas versus Normal Adult Tissues

R. Muraro, D. Wunderlich, A. Thor, J. Lundy, P. Noguchi, R. Cunningham, and J. Schlorn1

Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH [R. U., D. W., A. T., J. L, J. S.], and Office of Biologies, National Center for Drugs andBiologies, Food and Drug Administration [P. A/., R. C.], Bethesda, Maryland 20892

ABSTRACT

Monoclonal antibodies (MAbs) designated COL-1 through 15

have been generated and characterized and show a strongdegree of selective reactivity for human colon carcinomas versusnormal adult tissues. To prepare the MAbs, mice were immunized with extracts or membrane-enriched fractions of biopsy

material from either primary or metastatic colon carcinoma lesions. The fifteen COL MAbs, all of the ¡mmunoglobulin G subclasses 1, 2a, or 2b, reacted with purified Carcinoembryonicantigen in solid-phase radioimmunoassay and by immunoblot-

ting, but none reacted with human polymorphonuclear leukocytes or certain spleen preparations previously shown to be richin Carcinoembryonic antigen-related or cross-reactive antigens.MAbs COL-1 through 15 could be divided into at least five

groups based on their differential range of reactivities to thesurface of colon carcinoma cells or other carcinoma cell preparations. The COL MAbs were tested via immunoperoxidase witha wide range of primary and metastatic colon carcinomas, benignor dysplastic colon lesions, and 34 normal adult tissues. Withfew exceptions, the COL MAbs tested in this manner showedreactivity only to the primary and metastatic colon carcinomas.These studies thus provide a well-characterized repertoire of

MAbs that are well suited for potential clinical trials involving theradiolocalization and possibly therapy of human colon carcinomalesions. The fact that at least five epitopes are being recognizedalso provides the opportunity for testing the efficacy of cocktailsof the COL MAbs toward these goals.

INTRODUCTION

Several studies have identified colon tumor-associated anti

gens using both polyclonal antisera and monoclonal antibodies(1-8). One group of colon tumor markers has been identified asoncofetal antigens (1-4, 6, 7); these are defined as molecules

which are normally expressed during fetal development andreexpressed during neoplastic transformation. Of this class,CEA2 has been the most extensively studied (1,4,5,9-11). CEA

has been described as a M, 180,000 complex glycoprotein whichis highly expressed by both embryonic colonie mucosa and

1To whom requests for reprints should be addressed, at Laboratory of Tumor

Immunology and Biology, National Cancer Institute, NIH, Bldg. 10, Rm 8B07,Bethesda, MD 20892.

2The abbreviations used are: CEA, Carcinoembryonic antigen; MAb, monoclonal

antibody; PMN, polymorphonuclear leukocyte; RIA, radioimmunoassay; FACS,fluorescence-activated cell sorting; PBS, phosphate-buffered saline; SDS, sodiumdodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; NCA, nonspecific cross-reacting antigen; BSA, bovine serum albumin; G «MulgG, goat anti-mouse IgG;ABC, avidin-biotin complex; anti-HCEA, anti-carcinoembryonic antigen monoclonalantibody.

Received 12/18/84; revised 5/6/85; accepted 5/23/85.

carcinomas of the gastrointestinal tract. There have been numerous reports, however, that indicate that CEA may actuallybe a family of isoantigens and that anti-CEA antibodies may

indeed differ dramatically in their reactivity (12).There are several areas in which antibodies to CEA are being

used in the management of carcinoma patients; these include:(a) blood assays to monitor tumor burden; (b) immunohistopath-ological analyses of tissue samples to detect or further characterize tumor cells; (c) In situ detection of carcinomatous lesions(either at primary sites, regional lymph nodes, or distal sites)using radiolabeled antibody; and (d) potentially, therapy. One ofthe major drawbacks of the use of anti-CEA antibodies for theabove purposes has been the cross-reactivity of these reagentswith some apparently normal adult tissues, especially PMNs (13-20).

Previous studies have shown that most conventional hyper-immune antisera raised against CEA react with many differenttypes of carcinomas as well as with antigenic determinantsexpressed in normal tissues (13-16,21-25). Using purified CEA(11,17,18, 26-30), colon and gastric carcinoma cell lines (5,12,

20, 31, 32), and extracts of human breast carcinomas (8) asimmunogens, anti-CEA monoclonal antibodies have been generated previously which cross-react with CEA-related antigens

on normal colonie mucosa (18, 20, 26, 28, 29, 31, 32), normalspleen (20,28,33), normal liver (27,28), normal lung (28), sweatglands (33), polymorphonuclear leukocytes (17, 18, 20, 27, 28,32, 33), and monocytes (20). The CEA cross-reacting antigensmost commonly described are: NCA (13-16); NCA-2 (15,16,21,

23); normal fecal antigen (25); meconium antigen (19); and biliaryglycoprotein (24). At present, the degree of differential expression of CEA in carcinomas versus normal tissues is not resolved.A well-characterized group of anti-CEA MAbs (28, 34), some of

which are currently being used in radioimaging of colon cancerpatients, has been shown to have at least some degree ofreactivity to normal PMNs. Kuroki ef al. (30) have recently shownthat two MAbs reactive with CEA do not react with purified CEA-related determinants. These MAbs have not yet been characterized as to range of reactivity to tumor versus normal tissues,and they do not react with purified CEA preparations other thanthat used for immunization. Perhaps the most well-characterizedanti-CEA MAbs, in terms of range of reactivities to tumor and

nontumor tissues, are those recently reported by Primus et al.(28, 34) and designated NP-1 to -4. MAb NP-1 was shown toreact with polymorphonuclear leukocytes, and MAbs NP-1 to -3were shown to react with normal colon. The most highly selectiveanti-CEA MAb thus far reported in terms of differential reactivity

for colon carcinoma versus normal adult tissues is MAb NP-4.One interesting finding of these studies, however, was that the

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NP-4 epitope is principally expressed on primary colon carcino

mas and not in regional or distal métastases(34). Thus, with allthe studies reported characterizing numerous anti-CEA MAbs,to our knowledge none exist (with the exception of NP-4) thathave been extensively characterized as to differential reactivitywith primary and metastatic colon carcinomas versus benigncolon lesions and a wide range of normal adult tissues.

In this report we describe fifteen anti-CEA monoclonal anti

bodies, obtained from mice immunized with several differentpreparations from human colon carcinoma biopsy material.These MAbs were tested early in the screening processes forlack of reactivity to polymorphonuclear leukocytes. These MAbsare reactive with at least five epitopes on the CEA molecule, areshown to be highly selective for reactivity to colon carcinomasversus numerous normal adult cell types, and are siiown to bindto the surface of tumor cells.

MATERIALS AND METHODS

Cell Extracts and Membrane Preparation. The neoplastic and normalhuman adult tissues were obtained from Departments of Surgery andPathology, George Washington University, Washington, DC. Cell extractsand partially purified membranes of tissues and cell lines were preparedas described previously (35). Protein concentrations were determined bythe method of Lowry et al. (36).

Immunizations. Four-wk-old BALB/c mice were immunized by i.p.injection of 100 M9 of colon carcinoma cell extract or membrane-enrichedfraction, emulsified in an equal volume of complete Freund's adjuvant.

Seven days later mice were given an additional i.p. inoculation of 100 ^gof immunogen emulsified in an equal volume of incomplete Freund's

adjuvant. After an additional 7 days, mice were boosted i.v. with 10 ¿»gof immunogen, and spleens were removed for fusing 3 days later.

The immunogen used for the generation of MAbs CO1-9, -12, -13,and -15 was an extract of a human colon carcinoma metastasis to lymphnode. The immunogen used for the generation of MAb COL-2 was an

extract of a primary human colon carcinoma. The immunogen used forthe generation of MAbs COL-3, -5, -6, -7, -8, -10, and -14 was a partially

purified membrane preparation of the primary colon carcinoma used inthe above protocol. Extracts of two colon carcinoma métastases (onelymph node and one spleen) and an extract of a primary colon carcinomawere used as sequential immunogens for the generation of MAbs COL-1, -4, and -11. One hundred ^9 of the extract of the lymph node

metastasis were used as the primary immunogen followed in sequenceby immunizations with 100 ^g of the spleen metastasis and primarycarcinoma extracts. The final i.v. boost consisted of a mixture of 10 ngof each of both métastasesand the primary carcinoma extracts.

Hybridoma Methodology and Monoclonal Antibodies. Somatic cellhybrids were prepared using the method of Herzenberg (37) with somemodifications (35). All hybridoma cell lines were cloned twice by limitingdilution. All fifteen characterized hybridoma cultures were shown to benegative for the presence of Mycoplasma. Immunoglobulin isotypes weredetermined as described (35). A single pool of hybridoma tissue culturesupernatant from each MAb was used for all experiments describedbelow. Thus MAbs titer differences can be ruled out in the comparisonof one experiment to another. An anti-HCEA, an lgG1, was purchased

from Hybritech, Inc., La Jolla, CA. MAb B1.1, an lgG2a, is reactive withCEA and CEA-related determinants and was described previously (8).

Cells. The BALB/c nonsecreting myeloma cell line, P3-NS1-Ag4-1(NS-1 ), was obtained from Dr. J. Kim, NIH, Bethesda, MD. The WiDr andLS-174T colon carcinoma cell lines were obtained from the AmericanType Tissue Culture Collection, Rockville, MD. The HT-29 colon carcinoma cell line and breast carcinoma cell lines MCF-7 and BT-20 were

obtained from the Breast Cancer Task Force, National Cancer Institute,NIH, Bethesda, MD. The A204 rhabdomyosarcoma cell line and bladder

carcinoma cell lines HA-698 and HA-1054 were obtained from Dr. S.

Aaronson, National Cancer Institute, NIH, Bethesda, MD. Cell line WiDr-

51 is a subset of cells from the WiDr colon carcinoma cell line which wasderived by repeated fluorescence-activated cell sorting of WiDr with anti-

CEA MAb B1.1. All cell lines were maintained on the growth mediumrecommended by their respective sources. Purified CEA from five different colon cancer patients was provided by Dr. H. Hansen, Hoffman-La

Roche, Nutley, NJ. One CEA preparation was purchased from AaltoScientific, Ltd., San Marcos, CA.

Solid-Phase RIAs. Hybridoma supernatants were assayed for specificantibody production in solid-phase RIA using cell extracts from different

colon carcinomas, normal tissues, and purified CEA. Fifty //I containing5 ng of cell extract or 20 ng of purified CEA were added to each well of96-well polyvinyl microtiter plates and allowed to dry overnight at 37°C

in a nonhumidified incubator. The wells were quenched to minimizenonspecific protein binding by the addition of 100 n\ of 5% BSA in PBScontaining calcium and magnesium (PBS with Ca2+, Mg2+). The BSA wasaspirated after an incubation of 1 h at 37°C, and 50 n\ of tissue culture

supernatant were added to each well. After 1 h of incubation the unbound¡mmunoglobulin was removed, and the plates were washed with 1%BSA in PBS. Each well was then incubated for 1 h at 37°C with 125I-

labeled G «MulgG at 75,000 cpm in 25 ß\.The unbound G «MulgGwas aspirated, and the wells were washed extensively with PBS-1%

BSA. The wells were then subjected to autoradiography using KodakXAR film and DuPont Lightning Plus intensifying screens. The films weredeveloped after an overnight exposure at 70°C. The bound 125I-Ga

MulgG was also detected by cutting the individual wells and measuringthe radioactivity in a gamma counter.

Competition Solid-Phase Radioimmunoassay. Competitive binding

experiments were carried out to determine if the COL MAbs recognizedunrelated antigenic determinants. Each of the COL MAbs was tested forits ability to compete for the binding of 125l-labeled COL-1 to an extract

of LS-174T cells. Immunoglobulin of MAb COL-1 was purified from asciticfluid by Protein A-Sepharose affinity chromatography (38) and iodinatedfollowing the iodogen procedure (38). LS-174T extract (2.5 M9) was

adsorbed to each well of polyvinyl microtiter plates, and saturatingamounts of first (inhibitory) COL-MAb (50 ^I/well) were added. Afterincubation for 1 h at 37°Cand removal of unbound competitor antibodyby extensive washing, 125I-COL-1 (500,000 cpm/50 ^l) was added toeach well and incubated for an additional hour at 37°C. If two MAbs

recognize the same epitope, the bound inhibitory MAb will prevent thebinding of 125I-COL-1. Bound 125I-COL-1 was determined by cutting

individual wells and measuring radioactivity in a gamma counter, cpmbound to the wells preincubated with saturating COL-1 MAb as compet

itor was considered 100% competition.Fluorescence-Activated Cell Sorting. Flow cytometry target cells

were harvested and washed with Ca2*, Mg2+-free Dulbecco's PBS. Cells

(106) were incubated for 30 min at 4°Cwith 100 nl of hybridoma tissue

culture supernatant. After incubation, the cells were washed with complete medium and further incubated with 100 n\ of fluoresceinated goatanti-mouse ¡mmunoglobulin (Cappel Laboratories) for 30 min at 4°C.Thecells were then washed and suspended at 106 cells/ml in PBS with 2%

fetal bovine serum. The cell suspensions were analyzed in an OrthoCytofluorograf System 50 with an argon-ion laser tuned to 488 nm and

emitting 200 mW of power. Dead cells were gated out by axial extinction,and one-dimensional histograms were analyzed on an Ortho Model 2150

computer system. The percentage of positive cells was calculated bysetting a threshold of cells without primary antibody and subtracting theresulting value from the total percentage of antibody-stained cells.

Western Blotting. Forty ng of cell extracts or 1 «¿gof purified CEA,diluted in SDS-PAGE sample buffer [0.125 M Tris-HCI (pH 6.8)-4% SDS-20% glycerol-10% 2-mercaptoethanol], was loaded onto a 5 to 20%linear gradient SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose paper (0.45-Mm pore size) at 4°Cfor 4 h at 30 V

in transfer buffer [25 mw Tris-HCI (pH 8.3)-192 mw glycine-20% metha-

nol]. The blots were then incubated in PBS containing 5% BSA for 1 h

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TUMOR-ASSOCIATED CEA EPITOPES

at room temperature and washed with PBS containing 0.05% Tween-

20. Ten ml of hybridoma tissue culture supernatant were added, andincubation continued for 1 h at room temperature with gentle agitation.After washing with PBS containing 0.05% Tween-20, the blots wereincubated for 1 h at room temperature with 125l-labeled goat anti-mouse

IgG (Kirkegard & Perry, Gaithersburg, MD). The filters were then extensively washed overnight and exposed to Kodak XAR-5 X-ray film with aDuPont Lightning Plus intensifying screen at -70°C for 2 h. For all

experiments NS-1 tissue culture supernatant was used as a negative

control.Immunoperoxidase Studies. Reactivity of hybridoma tissue culture

supernatants with formalin-fixed 5-nm tissue sections was determinedusing a modification of the avidin-biotin-peroxidase complex (Vectastain

ABC kit; Vector Laboratories, Inc.) method (39). For each COL MAbtested, isotype identical control antibodies (lgG1: MOPC 21, LittonBionetics, lgG2a: UPC-10, Litton Bionetics) were used at identical con

centrations and conditions on parallel sections; no reactivity was ob

served.

RESULTS

BALB/c mice were immunized with extracts of human primaryand/or metastatic colon carcinoma following four different protocols as described in "Materials and Methods." Sequential im

munizations with three different tumors were also used in one ofthese protocols in an attempt to induce the production of antibodies to determinants shared by both metastatic and primarycolon lesions. Furthermore, cell extract and partially purifiedmembrane from the same primary colon carcinoma were usedin two different immunization protocols (see "Materials and Methods"). Splenocytes of immunized mice were fused with non-

immunoglobulin-secreting NS-1 myeloma cells to generate 5052

primary hybridoma cultures. Supernatant fluids from these cultures were harvested and tested in solid-phase RIA to detect the

presence of immunoglobulins reactive with extracts of theirrespective colon carcinoma immunogens and not with an extractof an apparently normal human spleen. The spleen used in theinitial screening was previously shown to be reactive with someMAbs that are cross-reactive with CEA and CEA-related deter

minants. Whereas immunoglobulins from many cultures demonstrated reactivity with both spleen and colon carcinoma extracts,341 cultures produced immunoglobulins reactive only with therespective colon carcinoma extract used. After double cloning ofhybridoma cells by limiting dilution, the resultant culture supernatants were assayed in solid-phase RIA for binding to extracts

of five colon carcinoma biopsy specimens and several apparentlynormal human tissues, including four spleens, two kidneys, andthree livers. Forty-nine MAbs were shown to be reactive with the

five colon carcinoma extracts and nonreactive with any of thenormal tissues. These MAbs were also shown to be nonreactivein solid-phase RIA with extracts of eleven preparations of RBC

representing all major blood groups (A, B, O), as well as withextracts of normal human PMNs. In addition, the forty-nine MAbswere tested using the ABC-immunoperoxidase technique for

reactivity to sections of five colon carcinomas and for lack ofreactivity to sections of normal human colon, sweat gland, bonemarrow, and lymph node. As a result of these initial screenings,fifteen MAbs, designated COL-1 to -15, were chosen for furthercharacterization. The reactivities in solid-phase RIA of MAbsCOL-1 to -15 with colon carcinoma extracts are shown in Table

1. None of the MAbs was reactive with extracts of biopsy material

from three normal adult livers and four normal adult spleens.Moreover all fifteen COL MAbs reacted in solid-phase RIA with

five preparations of CEA obtained from different colon cancerpatients. The differential reactivities of the MAbs to the coloncancer extracts and CEA preparations will be discussed below.All fifteen MAbs were of the IgG isotype of the 1, 2a, and 2bsubclasses as shown in Table 1.

MAbs COL-1 to -15 were then tested by FACS for binding to

the surface of normal human polymorphonuclear leukocytes; asdiscussed above, this has been the major cross-reactivity of anti-

CEA monoclonals and heterologous sera (14). The reactivities ofthese COL MAbs were compared to that of a commerciallyavailable anti-HCEA. As seen in Chart 1ß,the anti-HCEA MAb

reacted with the surface of over 90% of the human PMNs. Allfifteen COL MAbs were nonreactive with the same preparation;a representative example (using MAb COL-1 ) of this nonreactivity

to PMNs is shown in Chart ^A. As an extension of these studies,MAb COL-1 was compared with MAb B1.1 for reactivity with

various concentrations of an extract of PMNs. MAb B1.1 haspreviously been shown to be reactive with both purified CEA andCEA-related determinants (12). Using 1.6 ng of purified MAbsCOL-1 and B1.1, antigen dilution experiments revealed a positive

reactivity using MAb B1.1 with as little as 100 ng of PMNsextract. On the other hand, addition of as much as 5000 ng ofPMNs extract to reaction mixtures revealed no reactivity withMAb COL-1. Both B1.1 and COL-1 were equally positive for

reactivity to purified preparations of CEA.To further substantiate the results of Table 1 and Chart 1, an

extract of a normal spleen known to contain PMNs reactive withan anti-CEA MAb was tested for reactivity with the COL MAbs.As shown in Chart 1, inset, MAb COL-2 shows a high degree of

selective reactivity for a colon carcinoma extract versus theextract of this spleen.

MAbs COL-1 to -15 were then characterized as to their ability

to immunoreact with CEA in Western blotting experiments. Asseen in Fig. 1, the fifteen COL MAbs immunodetected the M,180,000 protein characteristic of the CEA molecule. As shownpreviously in Table 1, several patterns of differential reactivity tothe CEA preparations were observed. This finding is exemplifiedin Chart 2, A and ß;MAb COL-6 reacts equally with CEApreparations I and II (Fig. 2A), whereas MAb COL-1 reactspreferentially with the CEA-I preparation (Chart 2B). These re

sults provided preliminary evidence that the fifteen COL MAbsgenerated may be recognizing different epitopes within the CEAmolecule or among CEA molecules and have raised the possibility that these epitopes may be differentially expressed onvarious human tumor and/or normal tissues.

Previous studies (12) have shown that other monoclonal antibodies immunoreactive with the purified M, 180,000 CEA molecule may also react with M, 160,000, 90,000, 50,000, and/or40,000 proteins in colon cancer cells. These lower-molecular-weight molecules are believed to be the CEA cross-reacting

antigens that are expressed on polymorphonuclear leukocytesand other normal tissues. An example of this type of cross-

reactivity is shown in Fig. 26. Extracts from one colon metastasis, one primary colon carcinoma, and a normal spleen wereelectrophoretically separated in SDS-PAGE gradient gels and

transferred to nitrocellulose. After immunoblotting, MAb B1.1,previously shown to be reactive with CEA (8, 12), detected thecharacteristic M, 180,000 CEA molecule present in the colon

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Table 1Reactivity oímonoclonal antibodies in solid-phase radioimmunoassays

Cellextracts3Colon

carcinomaMAbCOL-1COL-2COL-3COL-4COL-5COL-6COL-7COL-8COL-9COL-1

0COL-11COL-12COL-13COL-14COL-1

5110,234"8,8157,40410,7371,40011,4298,8867,8853,4367,4409,0206,9704,5568,0835,799213,12313,3259.01912,7672,58912.82210,3389.2614,2259,16412,5767,8023.6348,1266,26839,4097.7698.4598,9111.1129.2989,0237,5113,3308,9229.0985,8274,1806.5454,66547,3166,7984,5026,5341.1656,9834,9163,1821,4704,4657,9513,7511,5314,7622,761513,93810,14810,22314,0791,89513,3911

1,3599,7354,6389.97810,2947,9755,4356,5266,230NormalLiver(n

=3)Neg8NegNegNegNegNegNegNegNegNegNegNegNegNegNegSpleen(n=4)NegNegNegNegNegNegNegNegNegNegNegNegNegNegNeg123,86224,1117,40628,4282,25613,84410,33311,4854,90811,33513,06111,1238,9808,84611,193219.81616,5876,16221.9871.67711.49311,6989,9437,1078,3619,9568,4618,1498,7768,558CEA"312,98915,1743,76026,2532,2259,7305,69310,4516,0705,1946,3367,5098,0512,4039,275424,27322,1398,62330,1173,43512,1019,98113,5168,6729,4497,9969,1115,74010,8809,963524,94825,8744,92031,0643,67715,41111,19512,0408,7606,86511,5749,2265,4409,60310,293lsotypeclgG2algG2bigGilgG2algG2blgG1IgGiigGilgG2bigGiigGilgG1lgG1lgG1lgG1

' Extracts prepared as described in "Materials and Methods."0 CEAs purified from five separate colon carcinoma patients.c Isotypes determined as described in "Materials and Methods."öcpmbound.9 Neg, negative; in all cases but one (binding of COL-4 to spleens, 693 cpm), the cpm bound was less than 500 cpm. Individualvalues are not given (three livers and

four spleens)for space considerations.

Chart 1. Flow cytometric analysis of anti-CEA MAbbinding to the surface of human polymorphonuclear leukocytes. Dashed lines in each panel represent the sortingpattern in the absence of primary antibody, i.e., negativecontrol A, sorting pattern (negative)using MAb COL-1; B,sorting pattern (positive) using anti-HCEA. Inset, the reactivity of COL-1 in solid-phase RIA versus', primary coloncarcinoma extract (•);normal spleen extract (•):PBS (A).

100

90

C) 80

f 70m 60

uj 50

F 40

3 30Å’ 20

10O

20 40 60 80 100 120 20 40 60 80 100 120 140

IMMUNOFLUORESCENCE

carcinoma extracts but also detected a M, 90,000 componentpresent in the primary carcinoma as well as the normal spleenextract. MAbs COL-1 to -15, on the other hand, detected only

the M, 180,000 CEA protein in extracts of colon carcinoma anddid not detect any other component in the same spleen extract.This is exemplified by the result shown in Fig. 2A.

Experiments were then conducted to determine if the fifteenCOL MAbs could be distinguished from one another on the basisof biological reactivities, i.e., on the basis of differential reactivities to a variety of colon carcinomas as well as carcinomas fromother sites. The fifteen COL MAbs were first tested by fluorescence-activated cell sorting for cell surface binding to the LS-

174T colon carcinoma cell line. As shown in Chart 3, all of thefifteen COL MAbs reacted appreciably to the surface of this cellline. However, upon testing for surface binding to two othercolon carcinoma cell lines (WiDr and HT-29), the fifteen COLMAbs displayed two separate patterns of reactivity. MAbs COL-1 to -5 bound similarly to the surface of all three colon carcinomacell lines, while the remaining ten MAbs (COL-6 to -15) displayedgreater surface binding to LS-174T cells when compared to

WiDr, and very little surface binding to HT-29 cells (Charts 3 and

4).An analysis of cell sorter histograms after the reactivity of the

fifteen COL MAbs to the surface of the WiDr-51 cell line revealeda dichotomy in antibody-mediated sorting patterns. The fiveMAbs (COL-1 to -5) that reacted equally to the surface of the

three colon carcinoma cell lines showed a Gaussian cell sorterpattern; this is exemplified in Chart 5A. The remaining MAbs,COL-6 to -15, which showed a differential reactivity to the surfaceof LS-174T, WiDr, and HT-29, could now be divided into twofurther groups. MAbs COL-6 to -12 showed a Gaussian cell-sorting pattern, exemplified in Chart 5B, while MAbs COL-13 to-15 showed a non-Gaussian cell-sorting pattern (see Chart 5C).

A Gaussian cell sort distribution (Chart 5, A and B) most probablyindicates a homogeneous expression of the reactive CEA epitopewithin the tumor cell population. The non-Gaussian cell sorter

pattern shown in Chart 5C most probably reflects a discretesubset of cells within the tumor cell population which expressthat particular CEA epitope only at a given time, thus demonstrating a heterogeneous distribution of the determinant. It

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TUMOR-ASSOCIATED CEA EPITOPES

10 -

Oen

O.o

24

20

16

12

o•o

03OCzO

1/LOG10 ANTIBODY DILUTION

100-,

80-

O

U)Oü_

60-

40

20-

0JCOL1 2345

E6b7D81]9D012113L14 1

ANTIBODYChart 3. Binding of MAbs COL-1 to -15 to the surface of three colon carcinoma

cell lines as analyzed by fluorescence-activated cell sorting. Results are expressedas the percentage of cells exhibiting fluorescence staining above that obtained inthe absence of primary antibody. •LS-174T; D, WiDr; D. HT-29.

should be pointed out that all fifteen COL MAbs were testedwith cells in the same growth conditions, and these results withdifferent MAbs cannot therefore be due to factors within cellcultures such as cell cycle. Thus the fifteen COL MAbs can nowbe divided into at least three groups on the basis of differentialreactivity to the surface of colon carcinoma cell populations (seeChart 4).

MAbs COL-1 to -15 were further characterized as to their

reactivities to other human neoplasms. Cell extracts of twohuman breast carcinoma cell lines MCF-7 and BT-20 and a cell

extract from a human breast metastatic lesion were assayed insolid-phase RIA for reactivity with the COL MAbs. As shown in

Table 2, thirteen of the fifteen COL MAbs reacted with theextract of the breast carcinoma métastases, but they did not

Surface binding toCoton cell lines in FACS.LS174T. WiDr. HT 29

Surface binding toWiDrSI in FACS(type of distribution)

Breast CaCell Eitr , SPRIA

Ino.pot/no.testedl

Bladder Ca

Ca«E«r . SPRIAIno pos no tested)

Rrabdomyo Sarc.Call E.tr . SPRIA(no poi'no tested)

5 MAbLS •TODr --HT29

10 MAHLS >WiDr >HT29

5 MAbGaussian

7 MAbGaussian

rL,

3 MAbNon Gaussian

2/2

I I

Chart 2. Differential reactivity of COL MAbs to CEA. Reactivity of MAb COL-6(A) and COL-1 (ß)with CEA preparations in solid-phase RIA. •,CEA preparation No MAb:

I; •CEA preparation II.MAb oaiignation: COL-1

COL-2

COL3COL-4

B

1

COL-5 COL-6COL-7

COLSCOL-9

COLIOCOL 11

COL 13COL 14COL-15

Chart 4. Categorization of COL MAbs 1 to 15 on the basis of differentialreactivity to carcinoma cells. SPRIA, solid-phase RIA.

react with extracts of the two breast tumor cell lines (see Table2 and Chart 6C). MAb COL-5 did not react to any of the threeextracts (Table 2; Chart 6A), while MAb COL-12 reacted to all

three (Table 2; Chart 6B). Thus the fifteen COL MAbs can nowbe divided into at least five groups on the basis of their differentialbinding to colon and breast cancer cells (Chart 4). The categorization of the fifteen COL MAbs into five groups was furthersubstantiated (i.e., did not change) by the reactivities of the COLMAbs to extracts of human bladder carcinoma cell lines (HA-1054, HA-698) and a rhabdomyosarcoma cell line (A204) (Table

2; Chart 4). The categorization of the COL MAbs into at leastfive groups based on their recognition of epitopes differentiallyexpressed on carcinoma cell lines cannot be explained on thebasis of relative antibody titers. For example, MAb COL-5 was

the only antibody which was negative for reactivity to all threebreast carcinoma extracts, but it was one of two MAbs thatscored positive to both bladder carcinomas. On the other hand,COL-12 was the only MAb which scored positive to all the

breast, bladder, and rhabdomyosarcoma extracts but reactedweakly to both the HT-29 and WiDr cell surfaces (Table 2; Chart

3). In all the above experiments, one lot of each MAb was usedfor all studies.

Competitive binding experiments were carried out to furtherdefine relations among the COL MAbs. MAb COL-1 was radio-labeled with 125Iand bound to an extract of LS 174T colon

carcinoma cells after preincubation with various amounts of theCOL MAbs. The COL MAbs used as competitors were representative of the five groups. MAb COL-1 (Group A, see Chart 4)competed completely as expected. MAbs COL-5 (Group B),COL-7 (Group C), COL-12 (Group D), and COL-15 (Group E) did

not compete using up to 125 times the relative amount ofantibody as COL-1. These findings substantiate those of Chart

4 that the COL MAbs define at least five CEA epitopes. MAb

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O 20 40 60 80 100 120 140 160 180 40 60 80 100 120 140 160 180

IMMUNOFLUORESCENCE

20 40 60 80 100 120 140 160 180 200

Chart 5. Flow cytometric analysis of MAb binding to the surface of WiDr-51 colon carcinoma cells. Dashed lines in each panel represent the sorting pattern in theabsence of primary antibody, i.e., negative control. A. MAb COL-1 ; B, MAb COL-6; C. MAb COL-15.

Table 2Reactivity of monoclonal antibodies to tumors other than colon in solid-phase

radioimmunoassayCell extracts3

Breast carcinomaBladder

carcinoma

Group MAb

Liver Rhabdomyo-MCF- BT metas- HA- HA- sarcoma

7 20 tases 1054 698 A204COL-1 Neg° Neg

COL-2 Neg NegCOL-3 Neg NegCOL-4 Neg Neg

11,25r 4,595 Neg Neg10,039 6,279 Neg Neg

5,762 2,534 Neg Neg15,406 4.547 Neg Neg

COL-5 Neg Neg Neg 3,552 2,519 Neg

C COL-6COL-7COL-8COL-9COL-1

0COL-11D

COL-12E

COL-13COL-14COL-1

5NegNegNegNegNegNeg1,719NegNegNegNegNegNegNegNegNeg2,691NegNegNeg11,1077,3207.4803,3985,2281

1,80414,0603,5485,3855,2632,7622,2391,9141,8781,4504,7643,3041,6641,2661,635NegNegNegNegNegNeg1,223NegNegNegNegNegNegNegNegNeg1,375NegNegNeg

a Cell extracts prepared as described in "Materials and Methods."0 Neg, negative, <1.000 cpm.c cpm bound.

COL-6 (Group C, see Chart 4) competed partially for the bindingof 125l-labeled COL-1. This finding may be the result of partial

cross-reactivity between the determinants recognized by COL-1and COL-6, or due to steric hindrance of the binding of the COL-1 MAb by the COL-6 MAb; i.e., the COL-1 and COL-6 determi

nants may be distinct but in close proximity. Our data favor thelatter view, since COL-1 and COL-6 can be clearly distinguished

on the basis of their differential reactivities to the surface of coloncarcinoma cell lines (Charts 3 and 5) and differential reactivitiesto purified CEAs (Chart 2). The biological distribution and competitive binding studies described here do not rule out thepossibility that more than five CEA epitopes are recognized bythe fifteen COL MAbs.

Immunohistochemical analyses were then used to determinethe range of reactivity of the COL MAbs to a wide variety ofmalignant and benign colon tumors, as well as to a wide rangeof normal adult tissues. The seven MAbs which best reacted ininitial screening with formalin-fixed sections of five colon carci-

ozom

o.U

13

- 12

2 -

1 -

I—I—T1 1 T

54321 54321 4321

1/LOG ANTIBODY DILUTION

Chart 6. Reactivity of MAbs to extracts of human breast carcinoma cells insolid-phase RIA; 5 i¡gof the following extracts were reacted with each MAb asdescribed in "Materials and Methods": A, MCF-7; •,BT-20; •metastatic breast

carcinoma. A MAb COL-1 ; B, MAb COL-12; C, MAb COL-9.

nomas were chosen for further study (COL-1, -4, -6, -9, -11,-12, and -14). As seen in Chart 7, the seven COL MAbs wereassayed against sections of between fourteen and twenty-threecolon carcinomas from different patients. For each of the MAbsa spectrum of reactivity was observed, ranging from no reactivityto the colon carcinoma cells of some patients to reactivity withgreater than 90% of carcinoma cells of carcinomas of otherpatients (Chart 7). The type of reactivity most often observed isshown in Fig. 3X\. Reactivity to the normal colonie epithelium inthe sections containing carcinoma was usually completely negative (Fig. 36) or consisted of a very weak reaction confined toonly a few epithelial cells.

Sections from ten benign colonie lesions of ten patients, representing several colonie disease states, were also assayed witheach of the seven COL MAbs. As seen in Chart 7, most lesionswere either negative or contained only a small percentage of thecells which were weakly reactive with the various MAbs. Ofparticular interest is the section of inflammatory bowel shown inFig. 3C. The seven COL MAbs tested were not only negative tothe colonie epithelium, but they were also negative to the adjacent PMNs and inflammatory cells, further substantiating thedata of Chart i A.

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10080Wö

60LUUOC200CO--

*•-••••••.-1VeoGODvoovco0.•••L-4V%00DOGOVCO••.:iL-6OODDO

WVCO.91AL-9OOffiCOL•A.ÌB-11DWCOL*»•••••-12doDODOvovCOL•••0ir.•••-14ADDDVOVA

ACC BENIGN ACC BENIGN ACC BENIGN ACC BENIGN ACC BENIGN ACC BENIGN ACC BENIGN

Chart 7. Immunoperoxidase staining of adenocarcinomas (ACC) of the colon and benign colon lesions. COL MAbs were reacted with sections of formalin-fixedparaffin-embedded human tissue using the ABC-immunoperoxidase technique. Each symbol indicates a tumor from a different patient. The percentage of positive cellsfor adenocarcinomas indicates the number of tumor cells scoring positive divided by the total number of tumor cells multiplied by 100. The percentage of positive cellsfor benign lesions denotes the number of epithelial cells positive divided by the total number of epithelial cells multiplied by 100 for each tumor or pathological lesion. •,primary colon adenocarcinomas. The following benign lesions are denoted by open symbols: O, tubulovillous adenoma; D, tubular adenoma; A, hamartomatous polyp;V. ulcerative colitis; O, diverticulosis; 6, chronic inflammation.

The reactivity of the seven COL MAbs was also studied andcharacterized using sections representing thirty-four normal adult

human tissues. As seen in Table 3, all seven MAbs were negativeto the vast majority of the normal tissues. MAbs COL-1 andCOL-4 (see Chart 4, Group A) both reacted with a few epithelial

cells from normal colon, stomach, and squamous oropharyngealmucosa (Table 3); these reactivities, moreover, were much lessintense (i.e., faint) as compared to reactivities with colon carcinomas. MAbs COL-6 and COL-11 (see Chart 4, Group C) were

negative to all normal tissues tested with the exception of aweak reactivity to approximately 1% of squamous epithelium ofthe skin (Table 3). MAb COL-9 (Chart 4, also Group C) demon

strated weak reactivity to approximately 1% of epithelial cells ofsmall intestine and 1% of renal tubule cells (Table 3). MAb COL-

14 (Chart 4, Group E) was strongly reactive with approximately100% of cells from the pancreatic islet of Langerhans (Table 3).This antibody was also reactive with a population of polymor-

phonuclear leukocytes histologically adjacent to colon carcinomacells and weakly reactive with some erythrocytes found in twoapparently normal organs; negative erythrocytes and PMNs wereobserved in the other normal organs tested (Table 3). MAb COL-

12 was negative with all the normal adult human tissues tested(Table 3; Fig. 3, D to G). It should be pointed out that a "negative"

reaction to normal adult tissue is only relative to the conditionsof the assay used in which each MAb scores strongly positiveto colon carcinomas. No claim is intended as to the absolutespecificity of a given MAb; the results presented simply demonstrate a differential reactivity to colon carcinomas versus normaltissues. Furthermore no fetal tissues have been examined.

A previous study has described a MAb generated using purified CEA as immunogen (28, 34). This MAb, designated NP-4, isperhaps the most selective anti-CEA MAb described in the

literature in that it shows no reactivity to normal tissues butreacts with primary colon carcinomas. This MAb, however, re

acted weakly or not at all to colon lymph node and distal colonmétastases(34). As seen in Table 4 and Fig. 3H, MAbs COL-4and COL-12 react similarly with colon métastasesas well as

with primary lesions. These data demonstrate that these twoMAbs recognize epitopes on the CEA molecule different fromthat recognized by MAb NP-4, and they further demonstrate the

wide range of reactivities that one may encounter among whathave been collectively termed "anti-CEA" antibodies.

DISCUSSION

Fifteen monoclonal antibodies (Col 1-15) have been generated

which recognize at least five epitopes on the CEA molecule andwhich discriminate primary and metastatic colon carcinomasfrom normal adult tissues. CEA, as originally described by Goldand Freedman (1), was defined as an immunoreactive glycopro-

tein found only in fetal and malignant gastrointestinal tissues.Recent studies, however, have demonstrated the immunologicalheterogeneity of CEA; polyclonal antisera and monoclonal antibodies raised against CEA also react with CEA-related antigens

present in normal adult tissues such as spleen, lung, colon,polymorphonuclear leukocytes, lymphocytes, and RBC (13-16,21-23, 25, 28, 34). Thus CEA has been further defined as afamily of "isoantigens" which have multiple antigenic determi

nants; some of these determinants may be common to all theisoantigens, and others may be antigenically distinct. BloodGroup A and nonspecific cross-reacting antigens, such as NCA,

may share one or more of these epitopes, but they are chemicallydistinguishable from CEA (13, 22).

Two complementary approaches were used for the studiesreported here in the generation and selection of MAbs: the firstwas the use of extracts of colon carcinoma biopsy specimensas immunogens to generate MAbs reactive with CEA; the secondapproach was the early and extensive screening of the MAbs

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Table 3

Reactivity of monoclonal antibodies to normal tissues using the immunoperoxidase technique

TissueMajor

organsLiverKidneyBladderLungTracheaBreastHeartGall

bladderSkinHematopoietic

tissuesBonemarrowLymphnodeSpleenThymusTonsilsPMN

neartumorRBCneartumorGastrointestinal

tissuesEsophagusStomachSmall

intestineColonNervous

tissuesNerve,peripheralBrain,cortexSpinal

cordCOL-1NegNegNegNegNegNegNegNeg10%"NegNegNegNegNegNegNegNeg1%"Neg1%"'CNegNegNegCOL-4NegNegNegNegNegNegNegNeg5%"NegNegNegNegNegNegNegNeg5%"Neg1%"'CNegNegNegCOL-6NegNegNegNegNegNegNegNeg<5%6NegNegNegNegNegNegNegNegNegNegNegNegNegNegCOL-9NegNeg8NegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNeg1%"NegNegNegNegCOL-1

1NegNegNegNegNegNegNegNeg1%6NegNegNegNegNegNegNegNegNegNegNegNegNegNegCOL-12NegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegCOL-14NegNegNegNegNegNegNegNegNegNegNegNegNegNeg1%"<1%"NegNegNegNegNegNegNeg

Genitourinary and reproductive tissues

ProstateTestesCervix, endocervixOvaryFallopian tube

Endocrine and exocrineglands

NegNegNegNegNeg

NegNegNegNegNeg

NegNegNegNegNeg

NegNegNegNegNeg

NegNegNegNegNeg

8 Weak staining of 1% of renal tubules6 Weak staining.0 Moderate staining of 5% of mucosal epithelium of appendix (focal within cytoplasm)." Intense staining of pancreatic islet cells.

NegNegNegNegNeg

NegNegNegNegNeg

ThyroidglandParathyroidglandAdrenalglandsPancreasSalivary

glandsMuscleSkeletal

muscleSmoothmuscleNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNegNeg100%NegNegNeg

Table 4Reactivity of MAb COL-4 with primary and metastat/c colon carcinoma using the

immunoperoxidase technique

TissueNo. of tissues

positivea/no. tested

PrimaryLymph node métastasesDistal métastases

15/1815/226/9

" A positive is scored as greater than 10% of tumor cells staining positive. Theintensity of staining was moderate and strong in all "positive" carcinoma lesions

and was comparable in primary tumors and regional and distal métastases.

for nonreactivity to those normal adult tissues, such as PMNs,that commonly expresses CEA cross-reactive antigens. Hy-bridoma cultures were not initially selected for their ability toproduce immunoglobulin reactive with purified CEA preparations.Rather, cultures synthesizing immunoglobulins reactive with colon carcinomas and not with a panel of normal tissues were

selected. In this way those cultures producing antibodies reactivewith normal tissues, regardless of anti-CEA reactivity, wereeliminated. The finding that the MAbs obtained were directedagainst these more selectively reacting CEA epitopes demonstrates that a range of CEA determinants exists that appears tobe peculiar to colon carcinoma versus adult normal tissue.

A major aim of this study was to generate MAbs that couldbe useful for radioimmunodetection of colon carcinomas and/ortherapy. For this reason it was not only necessary to generateMAbs displaying specificity for carcinoma cells versus normaltissues, but also that the MAbs bind an antigen expressed onthe surface of primary as well as metastatic colon cancer cells(see Chart 3). Immunizations for the generation of MAbs werecarried out with extracts and membranes of primary and metastatic human colon carcinoma biopsies as opposed to cell lines.The selective pressure in the establishment of colon cancer cell

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lines may eliminate or modify some antigens normally presenton human carcinomas. This has recently been demonstrated forat least one colon carcinoma-associated antigen (40). Thus extracts and membranes of primary and metastatic colon tumorswere used as immunogens.

Multiple assays including solid-phase RIAs with tumor ex

tracts, immunoperoxidase determinations on tissue sections,and FACS with live cells in culture have been used to reveal thediversity of the MAbs generated. As part of the screening process, the COL MAbs were also assayed for binding to the surfaceof normal polymorphonuclear leukocytes in FACS. While noneof the COL MAbs was reactive with the surface of these cells, acommercially available anti-CEA MAb displayed surface binding

to over 90% of the PMNs. This was not unexpected since amajor CEA-related antigen, termed NCA-1, is commonly ex

pressed by normal PMNs (14). These results may explain whyhigh levels of CEA are commonly detected by commerciallyavailable MAbs or antisera against CEA in the sera from manypatients with nonmalignant conditions (41) or smokers (41, 42).In fact, sera of patients with inflammatory disease could containimmunoreactive substances which mimic CEA in radioimmu-

noassay. Generation of monoclonal antibodies with restrictedspecificity to the different CEA isoantigens may be useful indefining more tumor-specific epitopes of CEA and may eventually

offer a more reliable serological diagnosis of cancer or detectionof tumor recurrence.

The immunohistochemical studies (Table 3) using the ABC-

immunoperoxidase method allowed the examination of a rangeof malignant and benign colon tissues as well as a wide rangeof normal adult tissues. Two MAbs (COL-1 and COL-4) did react

with small areas of normal colonie mucosa, but this reactivitywas focal and much less intense when compared to the stainingpattern on colon adenocarcinoma cells. The other COL MAbswere completely nonreactive with normal colonie mucosa. Theimmunohistochemical staining patterns observed (Table 3) supported the previous grouping of the MAbs (Chart 4). It should benoted that the immunohistochemical studies reported here utilized formalin-fixed (10% buffered) tissue sections. This was

done to facilitate the examination of a wide range of normaltissues as well as benign and malignant lesions. Further studiesusing frozen tissue sections should determine if the formalinfixation had any effect on reducing the binding of a given COLMAb to its reactive determinant. Preliminary studies indicate thatbinding to both some cells of normal colon as well as to carcinoma increased when several of the COL MAbs were used infrozen section.

Previous studies (28,34) have characterized an anti-CEA MAb,NP-4, which displays a similar selective reactivity to colon carcinomas versus normal tissues as the COL MAbs. NP-4, however,

does not react with distal métastases of colon carcinoma. Incontrast, at least two of the COL MAbs (COL-4 and COL-12)

were shown to react with the majority of the distal métastasestested. This suggests that NP-4 and COL-4 and COL-12 recog

nize different epitopes. In recent studies, Kuroki ef al. (30) haveshown that two MAbs directed against CEA do not react withCEA cross-reactive determinants. These MAbs, however, did

not react with purified CEA preparations other than that usedfor immunization. These determinants thus appear to be differentfrom those recognized by the COL MAbs described here (seeTable 1).

The COL MAbs have been divided into at least five differentgroups based on differential reactivities to carcinoma cells. Thishas been demonstrated by FACS using live cells in culture, solid-

phase RIAs to extracts of biopsy material and cell lines, andimmunoperoxidase assays to a range of tissue sections frombiopsy specimens. Each of the COL MAbs will now have to beevaluated for potential clinical utility. Recent studies by Ko-

prowski (43) and Bolognesi (44) have shown that MAbs of themurine lgG2a isotype elicit cell killing via macrophage mechanisms. MAbs COL-1 and COL-4 are of this isotype and will be

first evaluated for their ability to destroy human colon carcinomaxenografts in nude mice. The COL MAbs will subsequently alsobe evaluated for their ability to be (a) radiolabeled with isotope,such as 131I,and (b) reduced to Fab' and F(ab')2 fragments,

without loss of immunoreactivity. Those which can be bothsuccessfully radiolabeled and fragmented will be candidates foruse in in vivo detection and, using higher isotope levels, perhapsradioimmunotherapy of colon carcinoma lesions. Since at leastfive epitopes are being recognized by the COL MAbs, and notall cells of a given tumor express the epitope recognized by anyone COL MAb (Chart 7), subsequent in vivo or serological studiesusing mixtures of these MAbs are planned.

ACKNOWLEDGMENTS

We wish to thank D. J. Poole, M. Taylor, A. Sloan, and M. Ballard for experttechnical assistance in these studies. We also thank Dr. M. Weeks lor many helpfulsuggestions in the preparation of this manuscript and L. Otsby for photographicassistance.

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Fig. 1. Immunodetection of CEA by MAbs COL-1 to -15 using Western blotting analyses. Each lane represents the binding of a single COL MAb (COL-1 to -15) to aCEA preparation. Western blotting was performed as described in "Materials and Methods." Vertical scale, molecular weight markers (x 10~3).

Fig. 2. Immunodetection by Western blotting of CEA and CEA-related antigens in extracts of human tissues. Western blotting was performed as described in"Materials and Methods." Proteins detected by MAb COL-1 (A) and MAb B1.1 (B) in extract of: Lane 7, metastatic colon carcinoma: Lane 2, primary colon carcinoma;Lane 3. normal human spleen. Center scale, molecular weight markers (x 10~3).

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CANCER RESEARCH VOL. 45 NOVEMBER 1985

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TUMOR-ASSOCIATED CEA EPITOPES

B -

!

O

Fig. 3. Immunoperoxidase staining of paraffin-embedded fomialin-fixed human tissue sections using MAb COL-12. A, primary adenocarcinoma of the colon, invasivecomponent. The dark areas reflect the reaction of the diaminobenzidine substrate and thus MAb binding to cytoplasmic and membranous antigen in carcinoma cells. Thelighter areas are the hematoxylin countersign, i.e., no MAb reactivity, x 330. B, normal colonie mucosa. Normal colonie epithelium (arrow), stroma, and scatteredinflammatory cells are negative, x 130. C, tubulovillous adenoma (benign polyp); faint apical reactivity (arrow) with MAb COL-12. x 80. D, dermal sweat gland (sebaceoustype); nonreactive. x 130. £,normal lymph node follicle with a germinal center and mantle zone; nonreactive. x 330. F, normal bone marrow; nonreactive. x 540. G,normal liver. Hepatocytes, biliary epithelium and stroma nonreactive. x 330. H, adenocarcinoma of the colon metastatic to a lymph node. Reactive tumor (7), nonreactivelymphocytes (L), and lymph node capsule (c). x 130.

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1985;45:5769-5780. Cancer Res   R. Muraro, D. Wunderlich, A. Thor, et al.  

Normal Adult TissuesversusHuman Colon Carcinomas on Carcinoembryonic Antigen Differentially Expressed in Definition by Monoclonal Antibodies of a Repertoire of Epitopes

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