[CANCER RESEARCH 37, 2644-2650, August 1977]

SUMMARY

Antibody-dependent lymphocyte cytotoxicity against human colon carcinoma cells grown in vitro was demonshrahed with rabbit anli-carcinoembmyonic antigen (CEA)antisera and normal human lymphocytes. The same antisena produced no tumor cell lysis in a complement-dependent cytotoxicily test. The specificity of the reaction wasdemonstrated by the inhibition of antibody-dependent lymphocyte cytoloxicity after the addition of increasingamounts of purified CEA to the antiserum and by the factthat only tumor cell lines expressing CEA on their surfacewere lysed . Antibody-dependent lymphocyte cylotoxicihywas also observed against two colon carcinoma cell linesthat expressed Blood Group A antigen, using a humanserum containing anti-Blood Group A antibodies of the

immunoglobulin G class. This reaction was specifically inhibited by absorption with Blood Group A red cells, whereasthe anti-CEA-dependent cytotoxicity was not inhibited byabsorption with red cells of different blood groups.

INTRODUCTION

CEA3 was first identified by Gold and Freedman (14) inadenocancinomas of the digestive tract and in the digestiveorgans of human fetuses. Immunofluomescence studieshave shown that CEA is present on the surface of freshlydissociated colon carcinoma cells (15) and on establishedcolon carcinoma cell lines(4, 37). By immunoelechnonmicroscopy CEA is reported to be either localized in theglycocalyx of tumor cells (15) or closely associated with thecell membrane (18). Radioimmunoassay has shown thatCEA is released in the supennalant fluid of colon carcinomacellsgrown invitro(4,7,9,16,35).

The in vivo localization of radiolabeled anhi-CEA anhibodiesinhuman carcinomagraftedintohamsters(17)ornudemice (21) has been demonstrated. Thus it appeared impon

hant to determine whether antibodies against CEA might beactive in the destruction of tumor cells. Until now, however,only few results have been reported concerning the possible cylohoxic effect of anti-CEA antibodies toward coloncarcinoma cells (34).

In preliminary experiments with rabbit anhi-CEA antiseraand complement, we were unable to lyse a significant pencentage of colon carcinoma cells. Therefore the anti-CEAanhisenawere tested in the more sensitive ADCC assay (24,25, 29—31) against established colon carcinoma cell lines. Inthis assay a normal lymphoid cell population (K-cells) bearing a Fc receptor is capable of lysing antibody-coated targetcells (5). The results presented here demonslmate that rabbitantibodies directed against CEA can induce the specificlysis of tumor cells expressing CEA on their surface bynormal lymphocytes.

The relationship between CEA and blood group antigenshas been the subject of conflicting reports. Lo Gerfo et a!.(20) have shown that Blood Group A antigen can be presentas a contaminant of different CEA preparations, whereasGold et a!. (13) demonstrated that a Blood Group A-―like―antigen is linked to the CEA molecules. We found, however,that only some CEA preparations contained the BloodGroup A antigen whereas other CEA preparations can conlain Blood Group B on Lewis antigen depending on thegenetic background of the donor of the tumor from whichthe CEA was purified (19, 23).

In this report we also demonstrate ADCC by using humananti-Blood Group A antisera with tumor cells expressingthis antigen on their surface, thus showing that humanantibodies are as effective as are rabbit antibodies in ADCC.These latter experiments were designed not to determinethe exact relationship between CEA and blood group antigen but hodetermine the possible application of ADCC inanalysis of the specificity of 2 antigen-antibody systems byselective absorption of the respective antisera by the relevantantigen.

MATERIALS AND METHODSI Supported in part by Grant 3527075 from the Swiss National Foundation

for Scientific Research.2 To whom requests for reprints should be addressed, at Ludwig Institute

for Cancer Research, 1066 Epalinges s/ Lausanne, Switzerland.3 The abbreviations used are: CEA, carcinoembryonic antigen; ADCC,

antibody-dependent cell-mediated cytotoxicity; DMEM, Dulbecco's modifiedEagles medium; FCS, fetal calf serum; SR. spontaneous “Crrelease; CR,control ‘@Cr release; CDA, complement-dependent antibody cytotoxicity.

Received January 31, 1977; accepted May 13, 1977.

Purification of CEA.The preparationsof CEAusedfor theimmunization of rabbits and for the inhibition of the ADCCtest were purified from hepalic mehastases of colon carcinoma by 0.6 M perchlonic acid extraction, followed by Sephadex G-200and Sepharose6B (Phanmacia,Uppsala, Sweden) filtration as previously described (32).

CANCER RESEARCH VOL. 372644

Antibody-dependent Cell-mediated Cytolysis of Human ColonCarcinoma Cells Induced by Specific Antisera againstCarcinoembryonic Antigen1

S. Carrel,2M-C.Delisle, andJ-P. MachUnit of Human Cancer Immunology, Lausanne Branch, Ludwig Institute for Cancer Research (5. C., J-P. MI, and Department of Biochemistry, University ofLausannefM-C.D.J,1066Epalingess/ Lausanne,Switzerland

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ADCC of Colon Carcinoma Induced by Anti-CEA Sera

Anti-CEA Antisera and Anti-Blood Group A Antiserum. Antisera against purified CEA were prepared in nabbits. The animals received intradenmal injections of 200 j@gof purified CEA with complete Freund's adjuvant (DifcoLaboratories, Detroit, Mich.) into the hind fool pads, followed by booster injections of the same amount of antigenin the dorsal muscles every 15 days for 2 months.

Rabbit 110 was immunized with a CEA preparation codedCo-lOS (19) obtained from a Blood Group A patient; Rabbit142 was immunized with a CEA preparation coded Co-38(19) obtained from a Blood Group 0 donor.

For all ADCC experiments the anhi-CEA antisera wereabsorbed with perchlonic acid extract of normal lung (30mg/mI) in order to eliminate antibodies against the normalglycopnohein cross-reacting with CEA (NGP) (22) (NCA) (36).The specificity of the antisera was controlled by immunoelectrophoresis and by double diffusion. In control expeniments the anli-CEA antisera were also absorbed with BloodGroup A ABC or a pool of ABC from a different blood group.

Human anti-Blood Group A serum, obtained from a hypenimmunized donor (Gardner) was generously provided byDr. A. Holbumn (19).

All antisera were heat inactivated at 56°for 30 mm andstonedat—30°.

Purificationof Specific Anti-CEA AntIbodies. Specificanhi-CEA antibodies were isolated from rabbit antiserum byan immunoadsorbent column as previously described (21).The immunoadsorbent consisted of a copolymen betweenpurified CEA and bovine serum albumin, using 1% glutanaldehyde as a polymenizing agent. The adsorbed antibodieswere eluhed with 3 M NaSCN, dialyzed against 0.01 M TnisHCI buffer (pH 7.5), and concentrated to the original serumvolume. The activity of the antibodies was determined bytitration using a CEA radioimmunoassay.

Radloimmunoassayfor CEA. The liter of the anhi-CEAantisera and their inhibition by purified CEA was measuredby a double antibody nadioimmunoassay modified from themethod of Egan et a!. (8) as described by Fnilschéand Mach(ii).

Cells. Two human colon carcinoma cell lines were used:HT-29, originally established in culture by J. Fogh at theSloan Kettering Institute for Cancer Research (10); and Co115, originated in our own laboratories (4). Both cell lineswere routinely passaged as monolayer cultures and hamvested by incubation with 0.05% lmypsin:0.05% EDTA (Seromed, Munich, Germany) for 10 mm. The tumor cells weregrown in DMEM supplemented with 10% heal-inactivatedFCS (Gibco Bio-Cull Ltd., Glasgow, Scotland). The 2 celllines HT-29 and Co-i 15 have been shown ho express bothCEA and Blood Group A antigen on their surface (1, 4, 37).For control experiments 2 non-CEA-producing cell lines, ahuman endomelmial carcinoma, END-i (28), and a normallymphoblasloid cell line, LIK, were used. These 2 cell linesalso do not express the Blood Group A antigen.

51CrLabelingofTargetCells.Thecellswerelabeledwith51Cr by a modification of the method of Brunner et a!. (2).Target cells in suspension were adjusted ho 5 x 106/mI in0.15 M Tnis-phosphale-buffered saline containing 5% FCS.To 0.2 ml of the cell suspension (i0@cells), 100 @Ciof 51Cn(EidgenOssiches Inshilul für Aeaktorforschung , WLiren

lingen, Switzerland), with a specific activity of 200 @Ci/@gdiluted in 100 @l,were added. After incubationfon30 mm at37°with occasional gentle shaking, the labeled target cellswere washed 3 limes with 5 ml DMEM plus 5% FCS.

Preparationof Human Peripheral Blood Lymphocytes.Hepaninized venous blood (10 units/mI) was collected fromhealthy male donors. The blood was diluted 1:2 in culturemedium, and the lymphocyte-rich fraction was then separated on Ficoll:Hypaque (Seromed) gradients by centnifugalionat room temperaturefor 20 mm at 400 x g (Ficoll:sodium melnizoale; density, 1.077 g/ml).

ADCC.ADCCwas measuredby a short-term51Cnreleaseassay adapted from previously described methods (25, 30,31). All experiments were performed in duplicates or tniplicales that exhibited less than 5% variations. Briefly, i04labeled target cells in 20-pd volumes were distributed mbflat-bolbomed glass tubes and incubated with 20 p1 of theappropriate antiserum dilution for 30 mm at 37°beforeadding5 x io5lymphocytes inavolumeof 100 .d.The tubeswere centrifuged at 50 x g for 5 mm to increase contactbetween effechors and target cells and were incubated for 3hr at 37°in 5% CO2atmosphere. The ratio of lymphocytes tohanget cells was 50:1 in all experiments unless otherwisestaled. Al the end of the incubation period, 860 @lof coldmedium (DMEM) were added to each tube. The tubes werecentrifuged at 200 x g for 5 mm, and 500-@laliquols (50% ofthe final culture volume) were counted in a Packard gammascintillation counter. The following values were recorded:(a) the SR of the target cells incubated without antibody andlymphocyte; (b) CA of target cells incubated with lymphocytes and without antiserum ; (c) the test 51Cnrelease oftarget cells incubated with lymphocytes and antiserum; (d)the maximum 51Cnrelease (MA) obtained from 3-times frozen and thawed target cells in distilled water.

The percentage of specific 51Cnrelease was calculated byIhe following formula:

.. Av.TR—av.CR% specIfic 51Crrelease= x 100

Av. MR —av. CR

Inhibition of ADCC by Purified CEA or by RBC. Tworabbit anli-CEA anlisera (A-lb and A-142) that inducedstrong ADCC against HT-29 colon carcinoma cells wereabsorbed with increasing concentrations of purified CEA.After incubation al 37°for 60 mm and then at 4°for16 hr, thesera were centrifuged at 20,000 x g for 20 mm . The degreeof absorption was controlled in a titration curve of CEA inthe radioimmunoassay with a constant amount of 1251..labeled CEA and various dilutions of the absorbed antiserum samples. The CEA preparations used for these absomption experiments were not purified from the tumors of thesame patients who supplied the purified material used toimmunize the rabbits.

The human anti-Blood Group A serum, which was alsoactive in ADCC toward colon carcinoma cells HT-29 and Co115, was absorbed with various amounts of packed ABC ofBlood Group A on B donors with Ihe same incubation timesas described above for anli-CEA sera. In control expeniments, the anli-CEA antisera were also absorbed with ABCof different blood groups. Absorbed serum and antisera

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AntiserumdilutionIncubationtime (hr)SR°CRbSpecific

51Cr releasec1:500

1:10001241171:500

1:100034651461:500

1:10006121354 50theabsenceofperipheral blood

S. Carrel et a!.

samples were finally assayed in the ADCC test, the positivecontrols being the unabsonbed sera at the same dilutions.

CDA. CDA was measuredby the 51Crreleaseassaydescnibed by Brunneretal. (4). Briefly, i0@labeled target cellsin a 25-pivolume were distributedintoplastictubes and

incubated with 25 @.dof the appropriate antiserum on serumdilution for 30 mm at 37°,before 50 @.dof rabbit complementdiluted 1:2 were added. The rabbit complement was preabsorbed for 1 hr at 4°with 80 mg of agar (Bioquest, Cockeysville, Md.) pen ml in order ho abolish the spontaneous cylotoxicity of rabbit serum for human target cells. The activityof the absorbed rabbit complement was controlled usingrabbit anti-human species antiserum and HT-29 target cells.The tubes were then incubated for a further 3 hr at 37°.Calculation of the percentage of specific lysis was done asdescribed above for the ADCC test, taking as CA the figuresobtained by incubation of the target cells in the presence ofcomplement alone.

RESULTS

Anti-CEA Antibody-dependent Cell-mediated Cytotoxicity. In Experiment 1 , HT-29 cells derived from a colon canci

noma cell line known ho express CEA on its surface werelabeled with 51Cnand incubated for 30 mm with variousdilutions of 2 different rabbit anhi-CEA antisera (A-hO andA-142). Normal human peripheral blood lymphocyte from asingle donor were then added at a ratio of 50 lymphocyles/humor cell, and the cell mixture was further incubated for 3hr at 37°.Chart 1A shows that for the antiserum A-142 aspecific lysis of 60% was obtained at a dilution of 1:100 witha shill significant specific lysis (20%) up to a dilution of1:20,000. Similar results were obtained wihh antiserumA-hO except that there was a prozone effect observed athigh concentrations of antiserum. In this 3-hr experimentthe SR was 10% and the CA with lymphocytes was 15%,giving a 51Crrelease due to the lymphocytes in the absenceof antibody of 5% (CR-SA).

The kinetics of this ADCC reaction was studied in thefollowing way. Three series of tubes with labeled tumorcells containing different antiserum dilutions were incubated with lymphocytes from a single donor at a ratio of50:1 . At time intervals of 1, 3, and 6 hr, 1 duplicate of tubeswas collected. The results presentedin Table 1 showedthatthe specific lysis increased only slightly between 3 and 6 hrwhereas the CA with lymphocytes, which was usually low

@()r@@

@ .@

).

@:@@@ @()_.,.,,,,,,

Chart 1. A, percentage of specific lysis obtained with 2 rabbit anti-CEAantisera (R-110 and R-142) in a ADCC assay, using HT-29 colon carcinomacalls as target. B, percentage of specific lysis obtained with 2 rabbit antiCEA antisera (A-lb and R-142) in a ADCC assay, using Co-115 colon carcinoma cells as target.

Table1Influence of incubation time on ADCCusing anti-CEAantiserum

and HT-29 target cellsAnti-CEA antiserum used was R-142; effector:target cell ratio

was50:1.

a Target cells incubated in

lymphocytes and antiserum.b Target cells incubated in the presence of peripheral blood

lymphocytes and absence of antiserum.e Calculated according to the formula presented in “Materials

and Methods.―

between 1 and 3 hr of incubation, increased at 6 hr. Thespontaneous cyloloxic activity of the lymphocytes alone(incubated with uncoated target cells) varied in magnitudewith different lymphocyte donors. In order to minimize thisspontaneous cyhotoxiciby, all subsequent experiments weredone in 3 hr of incubation. In addition all tests giving a CRvalue at 3 hr higher than 20%were discarded. The level ofspecific lysis observed was dependent upon the number ofeffechor cells present up hoa lymphocyte:tumor cell ratio of50:1 , above which themewas no further increase in specificlysis. Therefore the ratio of 50:1 was adopted in all expenimenls.

The presence of K-cell activity in each lymphocyte preparation was controlled by coating the target cells with arabbit anti-human species antiserum (generously providedby Professor C. Bron). This antiserum has been found togive 40 to 60% specific lysis of all human target cells testedat a dilution of 1:2000 in the presence of active preparationsof lymphocytes. Experiments in which lymphocyte preparalionsgave lessthan40% valueswere discarded.

The pretreatment of target cells with 0.05% lrypsin:0.05%EDTA for 10 mm appeared to be the optimal procedure forthe preparation of target cells. In a comparative experimenttarget cells were prepared according to 3 different procedunes and tested simultaneously in an ADCC assay. Asshown in Table 2, the highest specific lysis was obtainedwith cells prepared with 0.05% tmypsin:0.05% EDTA, whilecells prepared either with a higher concentration of trypsin(0.25%) alone or with a higher concentration of EDTA(0.3%) alone gave significantly lower lytic values.

Colon carcinoma cells from another cell line (Co-uS),which expresses surface CEA (4), were also lysed in theADCC test by the 2 rabbit anti-CEA antisera A-hO and A-142. Chart lB shows that the specific lysis ranged from 45%at a dilution of 1:250 to 20% at a dilution of 1:10,000 for A-142 and 35 to 7% for the same dilutions of A-hO. Similarresults were obtained with 3 different rabbit anti-CEA antisara as well as with immunoadsorbent purified rabbit antiCEAantibodies. The maximum specific lysis obtained withthese different anhi-CEA antisera appears to be target celldependent. The values obtained with target cells from cell

.—., @@2

•_@@____.

‘N

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Pretreatmentoftarget cellsAntiserum dilutionSRbCRCSpecific

51Crrelease―0.05%

trypsin:0.05%EDTAfor10mm1:500

1:10006743380.3%EDTA for8mm1:500

1:1000121217120.25%trypsinfor10mm1:500

1:10006919 16

ADCC of Colon Carcinoma Induced by Anti-CEA Sera

3.4).The nadioimmunoassayconfirmed the progressive inhibition of the anli-CEA activity in the differenl absorbedantiserum samples, giving values ranging from 80% of binding with 125I-labeled CEA for the unabsorbed antiserum to12% of binding for the antiserum absorbed with 80 @gofCEAat a dilution of I :2000(Chart 3B).

Control absorption of rabbit anti-CEA A-hO and R-b42with packed ABC of different blood groups did not significantly influence the ADCC activity or the percentageof binding of ‘25I-labeledCEA. The results obtained afterabsorption of A-lb antiserum with 20% of Blood Group Apacked ABC are shown in Chart 2.

In order to test whether the inhibition of ADCC activity byaddition of purified CEA to the 2 anti-CEA antisera could bedue to CEA:anli-CEA immune complexes, the following expeniment was performed. Anti-CEA antisera absorbed with2 different CEAconcentrations were added to different dilu

Table2Influence of target cell preparation in ADCCa reactivity

(I Anti-CEA antiserum used was R-142; effectorto target cell ratio

was 50:1.

b Target cells incubated in the absence of peripheral blood

lymphocytesand antiserum.C Target cells incubated in the presence of peripheral blood

lymphocytesand absenceof antiserum.d Calculated according to the formula presented in “Materials

and Methods.―

line (HT-29) were always higher than those obtained withcells from the (Co-uS) line.

Controls with sena from nonimmunized rabbits and fromrabbits immunized with unrelated antigens gave no significant specific lysis at dilutions ranging from 1:5 to 1:5000.Other controls included the use of larget cells of 2 humancell lines, 1 endomelnial carcinoma line (END-i), and 1lymphoblastoid line (LIK), which have been shown by immunofluomescence not to express CEA on their surface. Withthese cell lines, no specific lysis was obtained at any dilulion of the 2 anti-CEA antisera A-hO and A-142, whereas inthe same experiment these cells were lysed by the rabbitanti-human species antiserum.

In order to muleout Ihal the lytic effect of anti-CEA antiserum could be due to antigen acquired by human cells growninFCS, thesame experimentswere performedwithtargetcells grown in 20% human AB@serum. The results obtainedusing cells grown in human serum were similar to thoseobservedwithcellsgrown inFCS.

Inhibition of ADCC by Purified CEA. Inhibition expeniments were undertaken to confirm that the ADCC activityobserved with the rabbit anti-CEA antisera was due to theinhemachionbetween the CEA and the corresponding antibody. Five hundred @.dof 2 rabbit anli-CEA antisera (A-lband R-l42) al a dilution of 1:2 were absorbed with 5, 10, 20,40, and 80 @igof purified CEA. The results obtained in theADCC assay with Ihese absorbed antisera samples are presented in Charts 2,4 and 3A. It can be seen that mosh ofthe ADCC reactivity was removed from antiserum A-i 10 byabsorption with as liblIe as 5 @gof CEA, with a neduclion toundetectable aclivity between 10 and 40 @gof absorbingCEA (Chart 2A). In parallel the percentage of binding of theabsorbed anli-CEA antisera with 1251-labeledCEA was determined in bihralion curves of radioimmunoassay (Chart 2B).

Similar results were obtained wihh the more potent antiCEA antiserum A-142. With this antiserum Ihe ADCC achivibywas progressively inhibited with increasing amounts of CEAused for absorption giving values of specific lysis rangingfrom 50% for the unabsorbed antiserum to 12% for theantiserum absorbed with 80 @gat a dilulion of 1:1000 (Chart

32 000

A

R-11O ADCC.—. PIOP4ABSORBED

@ ABS.20%RBC A70 £—A 5@@qCEA

0—0@ U—. 2Opq@ 60 0—0 40@.q@ +-+ 8Opq@

@350@ S

; @@sL::NA 40 N• U@: LL@Z@f

10

50 100 250 500 1000 2000 1000 2000 8000

RECIPROCAL ANTISERUM DILUTION

Chart 2. A, inhibition of the specific lysis obtained in the ADCC assaywith rabbit anti-CEA serum, R-110, by absorption with increasing amounts ofpurified CEA. Absorption of R-110 with 20% of packed Blood Group A RBC(ABC A) shows no inhibition of ADCC. B, percentage of binding of ‘@@llabeled CEA by the same rabbit anti-CEA antiserum samples, absorbed withincreasing amounts of purified CEA, used in A. Absorption of R-110 with20% of packed Blood Group A RBC (ABC A) shows no inhibition of bindingto “8I-IabeledCEA. RIA, radioimmunoassay.

70

@ 60

.@3@°

U

@ 40

@ 30z0

@ 20

10

Chart 3. A, inhibition of the specific lysis obtained in the ADCC assaywith rabbit anti-CEA serum , R-142, by absorption with increasing amounts ofpurified CEA. B, percentage of binding of ‘2I-IabeledCEA by the samerabbit anti-CEA antiserum samples, absorbed (ABS.) with increasingamounts of purified CEA, used in A. AlA, radioimmunoassay.

50 100 250 500 1000 2000 1000 2000 8000 16 000 32 000

RECIPROCALANTISERUM DILUTION

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Possiblerole of CEA:anti-CEAcomplexesin the inhibition oftheADCCreactionADCC

experiment performed on HT-29 cells; effectorcelI:targetcellratio, 50:1 ; incubation time, 3hr.SpecificCEA:anti-CEA

(immune 51CrreTest serum used complexes added)lease―R-68―1:2000

No461:8000No19R-681

:2000 R-1 10: 1 :500 absorbed48with40 /LgCEA1

:8000 R-1 10 1 :500 absorbed20with40 @LgCEAR-1101:500

No411:500absorbedwith40 No0@Lg

CEAR-681

:2000 R-142: 1:1000 absorbed45with80 @.LgCEA1:8000

R-142: 1:1000 absorbed21with80/LgCEAR-1421:1000

No451:1000 absorbed with No180

@gCEAto the formula presented in “Materials

S. Carrel et a!.

tions of a rabbit anti-human species serum of known activityin the ADCC assay. As shown in Table 3, addition of absorbed anti-CEA antisera, which may contain immune cornplexes, did not change the specific lysis of target cells in theADCC assay, confirming the specificity of the inhibition byabsorption with CEA.

Anti-BloodGroup A Antibody-dependentCytotoxicity.The effect of a human anti-Blood Group A serum from ahypenimmunized donor was also tested in the ADCC assayagainst the 2 carcinoma cell lines (HT-29 and Co-i 15) expressing both CEA and Blood Group A on their surface.Specific Iysis of the HT-29 cells ranging from 60% at adilution of 1:25 to 20% at a dilution of 1:2000 was achievedwith this antiserum (Chart 4). With Co-i15 as target cells,the values of specific lysis with the same antiserum werelower, giving 20% at a dilution of 1:100.

In control experiments, several normal human sera fromnonpolytmansfused blood donors of various blood grouptypes induced no ADCC against either target cell. Targetcells from END-b and LIK cell lines, which do not expressthe Blood Group A antigen, were not lysed in the ADCCassay by the anti-Blood Group A serum. The specificity ofthe reaction was controlled by absorption of the anti-BloodGroup A serum with 10 and 30% packed ABC of BloodGroup A and B. The results presented in Chart 4 show thatonly ABC of Blood Group A removed the ADCC activity ofthe serum, whereas the absorption with ABC of BloodGroup B did not affect the ADCC activity.

ANTI-BLOODGROUPA.—. NON ABSORBED

70@ 0—0 ABSORBED WITH 10% RBC B

‘U 0—0 ABSORBED WITH 30% RBC BIn

@ 60• ..-• ABSORBED WITH 10% RBC A

@ A—AABSORBEDWITH30% RBC A

g 5O@

U

@.@3O

@f: 1'@. _____ _____I II I@ I

25 50 100 200 500 1000 2000 5000 10.000

RECIPROCALSERUM DILUTIONChart 4. Inhibition of the specific lysis obtained in the ADCC assay with

a human anti-A serum, by absorption with RBC of Blood Groups A and B.

Table 3

2S SO ‘00 250 500 @000 2000 4000 8000 25 00 @O0 2@0 500 @000 2000 4000RECIPROCAL ANTISERUM DILU1ION

Chart 5. A, percentage of specific lysis obtained in the CDA and ADCCassay with 2 rabbit anti-CEA antisera (R-110 and R-142), using HT-29 coloncarcinoma cells as targets; B, percentage of specific lysis obtained In theCDA and ADCC assay with a human anti-A serum (Gardner), using HT-29colon carcinoma cells as targets.

CDA. For comparisonof ADCCwith CDA, labeledcoloncarcinoma cells (HT-29 and Co-b15) were coated in thesame experiment with rabbit anhi-CEA antisera (A-i 10 or A-142) and then tested after addition of either rabbit complement on human lymphocytes. These experimental condilions excluded the possibility that differences in antigenexpression on tumor cells related to culture conditionscould interfere in the results.The results presentedin Chart5A show that both anti-CEA antisera (A-i 1 0 and A-b 42) gave

no lysis of labeled HT-29 carcinoma cells in CDA even whenused undiluted, whereas in ADCC they gave a lysis similar tothat shown in Chart 1.

In contrast to these results with specific anti-CEA antiserum, where the CDA gave negative results and the ADCCgave a definite specific lysis, the anti-Blood Group A humanserum gave a specific lytic activity in both CDA and ADCC.Chart SB shows that in the CDA assay the percentage oflysis ranged from 70% at a dilution of 1:25 to 5% at a dilutionof 1:1000 whereas in the ADCC assay the percentage of lysisranged from 60% at a dilution of 1:25 ho 7% at a dilution of

a Calculated accordingand Methods.―

b R-68, rabbit anti-human species; R-110, rabbitanti-CEA; R-142,

rabbit anti-CEA.

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ADCC of Colon Carcinoma Induced by Anti-CEA Sera

1:4000. Both CDA and ADCC lyses were entirely abolishedby absorption with 10% of packed Blood Group A ABC andunchanged by absorption with the same amount of ABC ofB or0 bloodgroup.

DISCUSSION

The ADCC has been used ho show that rabbit antibodiesagainst CEA are able ho elicit the lysis of colon carcinomacells bearing CEA on their surface. Several control expeniments demonstrate the specificity of this anti-CEA anti bodydependent lysis. Target cells not expressing CEA on theirsurface, as determined by immunofluorescence, were notlysed. Absorption of the reactive antisera by purified CEAspecifically inhibited the ADCC activity. This absorption wascontrolledin a CEA radioimmunoassay, using as radioaclive tracer a CEA preparation different from the one used forabsorption. In addition, absorption of the anhi-CEA antiserawith ABC of different blood groups did not decrease theircytolytic activity, confirming that the activity was not due toa contaminating anti-blood group on other common membrane antigens such as /32-micnoglobulin. Finally, it wasshown that immunoadsorbent-punified anti-CEA antibodieswere also active in the ADCC assay. Although ADCC activitywas easily demonstrable up to very high dilutions of theanti-CEA antisera, the same antisera, absorbed with extractof normal tissue, were unable to produce any lytic activity inthe CDA, even at high concentration. These results suggestthat complement-dependent lysis may require a presenhalion or density of antigen on the target cell surface differentfrom that required for lymphocyte-dependent killing.

For example, the fact that CEA seems to be localized inthe glycocalyx (15) of the tumor cell and not in intimatecontact with the membrane could explain why anhi-CEAantibody and complement cannot lyse the target cellswhereas this antibody is capable of inducing sufficientlyclose contact between the attacking lymphocyte and thetargetcells.

Recent results not presented in this article showed that ananti-CEA antiserum of goat origin was not able to provokeany lysis of the colon carcinoma target cells in the ADCCassay despite the fact that the anti-CEA liter in the goatantiserum as determined in radioimmunoassay was muchhigher than that of the rabbit antisera. These negative mesuIts repeated even with immunoadsorbent-pumified goalanti-CEA antibodies suggest that human K-cells may beunable ho recognize the Fc of a goat lgG.

The recently published results of Tompkins et a!. (34)differ from our own observations in that these authors oblain a specific lysis of HT-29 target cells in CDA with goatanti-CEA antisera. Two explanations can be given for thediscrepancy between the results: (a) the group of Tompkinsused a highly toxic rabbit complement yielding a nonspecific lysis of 42% with HT-29 targets, whereas we used arabbit complement absorbed with agar, giving no additionallysis as compared with the SR of target cells alone; (b) inour hands, rabbit and goat anti-CEA antisera when usedunabsorbed also gave relatively high percentages of lysis onHT-29 target cells in CDA (ranging from 70 ho 10% at dilulions of 1:2 ho 1:64). However, this CDA lysis was abolished

by absorption of the antisera with penchlonic acid extractsfrom normal lung tissue. This absorption did not decreasethe anti-CEA titer of our antisera as controlled by nadioimmunoassay, and the absorbed rabbit antisera gave theADCC specific lysis reported in this paper. These resultsillustrate the importance of using anhi-CEA antisera of conbroIled specificity.

With anti-Blood Group A serum, the difference betweenADCC and CDA lysis is less striking. The high lytic activityobserved in CDA may be due to the fact that the human antiBlood Group A serum contained in addition to IgG a relalively large amount of 1gM anti-A antibody. A quantitativedifference in lytic activity between the CDA and ADCC achyity is, however, clearly demonstrated at high dilutions of thisserum.

It is attractive to speculate that if a patient had lgG antiCEA antibody in his serum his lymphocytes could kill someof his colon carcinoma cells by the ADCC mechanism.However, there are several arguments against this hypothesis. First the presence of lgG anti-CEA antibody in patientsera has never been demonstrated . In the majority of casesreported by Gold et a!. (12), where antibody could be detected by nadioimmunoeleclrophoresis against an allogeneic CEA preparation, these antibodies were found to bedirected against a “BloodGroup A-like―antigen, associatedwith the CEA preparation (13). In addition, in the few caseswhere authentic anhi-CEA antibody seemed to be presentthey were of the 1gM class (12). Other investigators havebeen unable to detect authentic anhi-CEA antibody in patient's sera (6, 20, 26, 33).

One could envisage that immunization with chemicallymodified CEA and adjuvant could break the natural humantolerance against CEA. However, even allowing the hypohhesis that this tolerance could be successfully broken, thefact that CEA is not exclusively present on colon carcinomacells but also in smaller concentrations in normal organssuch ascolon,lung,and breast(3,ii,27,32)makes itUnlikely that active or passive CEA-mediated immunotherapycould be of practicalvalueto helpthe colon carcinomapatient.

ACKNOWLEDGMENTS

We are grateful for the excellent technical assistance of S. Salvi and M-F.Hamou in these studies. We thank Dr. A. Campbell and Professor J-C.Cerottini for suggestions and advice.

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CANCER RESEARCH VOL. 372650

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1977;37:2644-2650. Cancer Res   S. Carrel, M-C. Delisle and J-P. Mach  Carcinoembryonic AntigenCarcinoma Cells Induced by Specific Antisera against Antibody-dependent Cell-mediated Cytolysis of Human Colon

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