[CANCER RESEARCH 34, 2159 2164, September 1974]

Affinity Cytotoxicity of Tumor Cells with Antibody-Glucose OxidaseConjugates, Peroxidase, and Arsphenamine1

Gordon W. Philpott, Richard J. Bower, Keith L. Parker, William T. Shearer,and Charles W. Parker

Departments of Surgery [G. W. P., R. J. B,] and Medicine [K. L. P., W. T. S., C. W. P.], Washington University School of Medicine, St. Louis,Missouri 63110

SUMMARY

Selective cytotoxicity was accomplished in two antibody-tumor cell models with antibody-glucose oxidase conjugates(Ab-GO) followed by treatment with horseradish peroxi-dase (HRP) and arsphenamine. The two cell models usedwere hapten [2,4,6-trinitrophenyl (TNP)]-substitutedHeLa and HEp-2 cells with specifically purified antihapten(TNP) antibody and human colonie carcinoma cells(HT-29) with immunoglobulin G anti-carcinoembryonicantigen antibody. Brief treatment of TNP cells with anti-TNP antibody conjugated to glucose oxidase (0.07 to 70/ig/ml) followed by culture in medium with HRP (50fig/ml) and arsphenamine ( 1to 10/ng/ml) resulted in zero to100% cell killing when compared with controls in a mi-crocytotoxicity assay. Cytotoxicity was reduced or absentwhen (a) any of the three components (Ab-GO, HRP, orarsphenamine) were omitted; (b) cells not substituted withTNP were used; or (c) free hapten (dinitrophenyl-lysine)inhibited Ab-GO binding to TNP cells. Affinity cytotoxicity (73 to 90%) was also seen in HT-29 cells treated withanti-CEA antibody conjugated to glucose oxidase followedby treatment in HRP and arsphenamine (1 to 10 jug/ml).CEA, extracted from malignant ascitic fluid with perchloricacid, partially inhibited the cytotoxic action of the Ab-GOsystem, and normal goat immunoglobulin G-glucose oxidase caused significantly less killing, showing the selectivityof the reaction.

INTRODUCTION

Selective tumor cell destruction can be improved byattaching potent toxins to tumor-specific antibodies, so thatthe cytotoxin adds killing potential to the antibody and theantibody gives tumor specificity to the toxin. This approach,which was proposed by Ehrlich many years ago (5), hasrecently proved to be effective with several antibody conjugate systems in experimental models (6, 13, 14, 16, 18-21).We have extended this approach to enzymes that arecapable of converting protoxins to toxins and have shown ina number of cell models that antibody conjugated to glucoseoxidase, an enzyme that generates H2O2 in the presence ofglucose, will selectively iodinate and kill cells when lac-

1Supported by Grant 5R01-CAI2626 from the National CancerInstitute.

Received March 7, 1974; accepted May 6, 1974.

toperoxidase and iodide are added to the system (18 21).The cell damage is produced by a catalytic iodination of thecell membrane by lactoperoxidase with H2O2 serving as thedriving force in the reaction. There are a number of otherpotentially potent oxidizable substrates that could utilizeperoxidase and the hydrogen peroxide that is locallyliberated by the Ab-GO.2 During the course of systematicscreening studies, we have found that arsphenamine, apotent cytocidal agent once used in the treatment ofsyphilis, is activated by HRP in the presence of glucoseoxidase and glucose.

The basic reactions in this system are as follows:

Glucose glucoseoxidase gluconic acid + H2O2 (A)

NH2

peroxidaseH202 + HO " " •¿� •¿�" » --- '

Arsphenamine

In the present study this system has proved to be highlyeffective in promoting selective cytotoxicity in 2 differentantibody-tumor cell models, namely, (a) hapten-substitutedHeLa and HEp-2 cells using specifically purified antihaptenantibody, and (b) human colonie carcinoma cells usingantibody directed to CEA (7).

MATERIALS AND METHODS

HeLa and HEp-2 cells (American Type Culture Collection, Rockville, Md.) were maintained at 37°in a humidified 5% CO2-95% air atmosphere in Eagle's minimal

essential medium with 10% PCS (Grand Island BiologicalCo., New York, N. Y.). HT-29, a cell line originally derivedfrom a human colonie adenocarcinoma and known to have

2The abbreviations used are: Ab-GO, antibody-glucose oxidase conjugate; HRP, horseradish peroxidase; CEA, carcinoembryonic antigen; PCS,fetal calf serum: HT-29, cell line derived from human adenocarcinoma ofcolon; TNP, 2,4,6-trinitrophenyl; PBS, phosphate-buffered saline;TNP-GO, anti-TNP-glucose oxidase conjugate; IgG, IS immunoglobulinG fraction; IgG-GO, IgG-glucose oxidase conjugate; a-CEA-GO, goatanti-CEA antibody-glucose oxidase conjugate; n-GS-GO, normal goatserum (IgG fraction)-glucose oxidase conjugate.

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significant amounts of CEA (4), was originally supplied byDr. J. Fogh (Sloan-Kettering Institute, Rye, N. Y.) andcultured at 37°in 10% CO2-90% air in McCoy's Medium5A with 15% FCS. All FCS's were heat inactivated at 56°

for 30 min, and all media contained penicillin (100 units/ml)and streptomycin (100 ¿¿g/ml).Single-cell suspensions wereobtained for HeLa and HEp-2 using 0.05% trypsin digestionfor 10 min and for HT-29 using EDTA (20 Mg/ml) for 10min and 0.35% trypsin for 10 min in balanced salt solutionwithout CA++ or Mg+*.

Hapten-substituted Cell Model. The methods for obtaining viable hapten-substituted cells and specifically purifiedantihapten antibody have been described in detail (18).Briefly, TNP-substituted HeLa or HEp-2 cells were obtained by incubation of PBS-washed cells (2 to 4 x IO6)with TNP-sulfonic acid (50 /ig/ml) in balanced salt solution for 20 min after which unbound TNP-sulfonic acid wasremoved with 2 washes of minimal essential medium plus10% FCS. Unsubstituted control cells were treated in anidentical fashion except that TNP-sulfonic acid wasomitted. A high-affinity anti-TNP antibody was purified(over 92% pure antibody) from the sera of rabbits hyper-immunized with TNP-bovine -y-globulin and completeFreund's adjuvant (Difco Laboratories, Inc., Detroit,

Mich.) by the method of Little and Eisen (11).Anti-TNP antibody was conjugated to glucose oxidase

(Boehringer Mannheim, New York, N. Y.) using a bifunc-tional imidoester, diethylmalonimidate, according to amodification of the method of Dutton et al. (3), aspreviously described in detail (21). This conjugate (anti-TNP-GO) contained about I molecule of glucose oxidaseper 10 molecules of antibody.

Preparation of A-CEA-GO. CEA, extracted from humancolonie adenocarcinoma by the method of Krupey et al. (9),was used to raise anti-CEA antibodies in goats immunizedwith multiple injections of CEA in complete Freund's

adjuvant. Antiserum was found to have a high titer ofanti-CEA antibody (significant binding of 125I-labeledCEAat a dilution of 1:10,000) when analyzed against purifiedreference CEA (kindly supplied by Dr. Charles Todd, Cityof Hope, Calif.) using agar gel diffusion, Ouchterlony, andradioimmunoassay techniques (1). The IgG from this anti-serum was isolated by precipitation with ammonium sulfateat 40% saturation, and DEAE-cellulose chromotography(17). The lyophilized IgG fraction was stored at -20°until

conjugated to glucose oxidase by the bifunctional imidoestertechnique (3, 21). For this 12.8 mg of IgG protein and 26 mgof glucose oxidase were allowed to react with diethylmalonimidate in sodium borate buffer, and the IgG-GOconjugate was precipitated and washed with ammoniumsulfate at 42% saturation. Then the IgG-GO was dissolvedin PBS and dialyzed extensively against the same buffer.This yielded 10.6 mg of IgG-GO based on absorbance at280 and 412 nm. n-GS-GO was prepared in the same wayfrom serum of unimmunized goats. Glucose oxidase activity, as determined by a fiuorometric assay (8), was similar for equal concentrations of the 2 conjugates.

To demonstrate specific inhibition of a-CEA-GO activity,CEA was partially purified by the method of Krupey et al.

(9) using perchloric acid extraction of ascitic fluid obtainedfrom a patient with widespread peritoneal adenocarcinomaof the colon. Using a radioimmunoassay with CEA provided by C. W. Todd as a reference, 1 mg (by weight) of theextracted ascitic fluid was found to contain 270 ng of CEA.In the selective cytoxicity studies with a-CEA-GO (Table 5),this partially purified CEA was added to cells just prior tothe conjugates and was present throughout the 30-minincubation period.

Preparation of Arsphenamine and Peroxidase. For eachexperiment HRP (type II, Sigma Chemical Co., St. Louis,Mo.) was freshly dissolved in PBS (I mg/ml) and sterilizedby filtration. Arsphenamine hydrochloride was synthesizedby the method of Christiansen (2). The product had amelting point of 190 197°(literature, 180 195°).Stock1-mg/ml solutions of arsphenamine hydrochloride in waterwere prepared, filtered, stored at -20°,and used within 2 to

21 days. A few min before an experiment, 10 to 100 ¿¿1ofstock solution were added to 1 ml of nutrient medium. Thefinal pH of the medium remained at 7.2 to 7.4.

Assay of Cytotoxicity. All experiments were initiated bywashing single cells (1 to 2 x 10") with PBS and adding theappropriate amounts of Ab-GO. After incubation for 30min with gentle agitation, unbound Ab-GO was removedwith 2 washes, and the cells were counted in a hemacytome-ter and distributed into appropriate media with or withoutHRP and arsphenamine. For all control and experimentalconditions a known number of cells (500 to 1000) waspipetted into microtest wells (Falcon Plastics Co., Oxnard,Calif.). After 18 to 20 hr incubation, the plates were fixed,stained, and scored for viable cells in a standard area by themicrocytotoxicity technique previously described (18, 19).Six to 18 wells were averaged for each determination.Cytotoxicity (percentage of cell kill) was calculated for eachsample as the average number of control cells counted perwell minus the experimental cells per well divided by controlcells. Mean Cytotoxicity values were calculated from 2 to 4samples.

RESULTS

To determine whether arsphenamine is subject to oxidation by HRP, spectrophotometric studies were carried outon the presumption that one of the major oxidation

products would be O=\CJ)/ As=o, which would have

significant absorbance in the 300 to 450 nm range. Preliminary screening studies indicated that when HRP was presentand supplied with a source of H2O2 (glucose oxidase andglucose), solutions of arsphenamine rapidly darkened anddeveloped an absorption maximum at 400 nm. Moredetailed studies (Table 1) established that the rate of thespectrophotometric change was dependent on the concentration of both HRP and glucose oxidase with little or nochange in the absence of glucose. Thus, strong presumptive

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Table 1Enzymatic oxidation of arsphenamine

A 50 ¿IMsolution of arsphenamine in 0.1 Mphosphate, pH 7.4 (freshlyprepared), was placed in a Beckman cuvet at 24°in the presence or absenceof 5 IBMD-glucose and HRP, 0.01, 0.03, 0.13, or 0.33 unit/ml. The reactionwas initiated by the addition of 0.01 volume of glucose oxidase (giving afinal glucose oxidase concentration of 0.1 or 1.0 unit/ml). Serial readingswere made at 400 nm, the absorption maximum of the arsphenamineoxidation product(s). Timed 400 nm readings in the absence of bothenzymes ranged between 0.05 and 0.07.

Condition1234567gGlucoseoxidase(units/ml)1.01.01.01.01.00.10.10.1HRP

Glucose,(units/1mg/ml)

ml0.33+0.33

+0.13+0.03+0.13+0.03+0.01

+Absorbance

at 400nm1

min0.0690.0920.4020.3080.0540.0500.0680.0866min0.0550.0720.4390.4720.1490.3370.1420.09012min0.0540.0680.4450.4920.2270.5690.3590.108

evidence for enzymatic oxidation of the arsphenaminemolecule was obtained.

Selective cell kill was obtained in both antibody-tumorcell models with the antibody-enzyme arsphenamine system(Tables 2 to 5). In the complete system of anti-TNP-GO,arsphenamine, and peroxidase, TNP cells showed up to100% killing whereas unsubstituted HeLa and HEp-2 cellswere much less cytotoxic (Table 2). When any of the 3components (antibody, peroxidase, or drug) were left out,the degree of TNP cell killing was markedly reduced orabsent. At the higher levels of arsphenamine (10 /¿g/ml),

Selective Cytotoxicity with Antibody Conjugates

some nonspecific cytotoxicity was seen that did not requireconjugate and was as marked in unsubstituted as in TNP-substituted cells (Table 2). The toxicity of arsphenamine iswell known, and it is not surprising that there is some degreeof nonspecific toxicity. In every case, however, the bindingof glucose oxidase to the cells by means of the antibody conjugate greatly increased the degree of cytotoxicity in TNPcells (Table 2). This was also true in the HT-29 cells (seebelow).

It can be assumed that the degree of anti-TNP-GObinding is dose dependent, since the level of specificcytotoxicity (above that seen with arsphenamine and HRPalone) varied markedly with initial antibody concentration(Tables 2 to 4). Significant antibody-dependent cell killingwas seen with as little as 1^g of Ab-GO per ml and reacheda maximum (100%) with about 20 ng/m\ (Table 3).

The specificity of anti-TNP antibody reaction was furtherdemonstrated by hapten inhibition of TNP cell killing in thecomplete system (Table 3). The presence of hapten (dini-trophenyl-lysine) during treatment of TNP cells with anti-TNP-GO caused a suppression of selective cytotoxicityafter HRP and arsphenamine were added. Thus, whenanti-TNP-GO binding was minimized or suppressed eitherby using cells without TNP (Table 2) or hapten inhibition inTNP cells (Table 3), cytotoxicity was much reduced,demonstrating the antibody specificity of the reaction.

This enzyme conjugate system was also effective inselectively killing colonie carcinoma cells in vitro (Table 5).In the HT-29 cells, the a-CEA-GO treatment caused amarked degree of cell killing (73 to 90%) after exposure toarsphenamine (1 to 10/ig/ml) and peroxidase. At the lowerlevel of arsphenamine (1 Mg/m'), the n-GS-GO caused littleor no significant cytotoxicity (in the complete system),

Table 2Selective cytotoxicity of TNP cells with anti-TNP-GO, peroxidase, and arsphenamine

All cells were incubated with Ab-GO or PBS for 30 min, washed 2 times with PBS, and culturedin minimal essential medium + 15% heated PCS with or without arsphenamine and HRP.Cytotoxicity was determined by the microcytotoxicity assay 20 hr later using the group not treatedwith Ab-GO, arsphenamine, or HRP as control. The values are the mean cell kill calculated from 2,3, or 4 samples. Average number of cells counted per well (total 6 to 12 wells) in the control groupswere 98.4 ±9.1 for TNP-HeLa; 89.7 ±6.0 for HeLa; 88.0 ±8.9 for TNP-HEp-2; and 104.5 ±6.9for HEp-2. Standard errors for other values ranged from 2.4 to 18.5.

Conditions Cytotoxicity (%)"

Ab-GO(Mg/ml)00005656565656Arsphenamine(Mg/ml)01-105100101510Peroxi

dase(iig/ml)50050500-500505050TNP-HeLa40-17"029«0-928"N.D.32"100"HeLa07N.D.C27"1713N.D.N.D.36"TNP-HEp-240-111321"0-13N.D.29"74"100"HEp-206-119N.D.017N.D.30'N.D.

"Standard errors less than 10% for all values.*Significantly different (p < 0.05) compared with control value (nutrient medium alone).CN.D., not done." Significantly different (p < 0.001) compared with control value (nutrient medium alone).

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G. W. Philpott et al.

Table 3Hapten inhibition of selective cytotoxicity of TNP cells

TNP-HEp-2 cells (5 x 10s) were incubated with or without Ab-GO inthe presence or absence of hapten (dinitrophenyllysine, 100 ¿IM).Afterwashing, cells were plated with McCoy's Medium 5A plus 15% heated

PCS containing arsphenamine (10/¿g/ml)and HRP (50/ig/ml)and scored20 hr later by the microcytotoxicity assay using the group without Ab-GOas control. The average number of cells counted per well (total 18wells) inthe control group was 93.4 ±8.2. Standard errors for the other groupsranged from 3.8 to 8.9.

Conditions Cytotoxicity (%)"

Arsphen-Ab-GOaminePeroxidase(jig/ml)

(lO^g/ml)(SOfig/ml)22

++11++1++0+ +Withouthapten100"89'23'Withhapten56»42»150

" Standard errors less than 10% for all values."Significantly different (p < 0.001) from control (without Ab-GO).' Significantly different ( < 0.05) from control (without Ab-GO).

Table 4Cytotoxicity with varying concentrations of Anli-TNP-GO conjugate

Procedures were the same as in Table 1 except the arsphenamine wasfreshly mixed and used within 1 to 2 hr. of dissolving in H2O. The averagenumber of cells counted per well (total 12 wells) in the control group(without Ab-GO, arsphenamine, or HRP) was 65.8 ±9.7. Standard errorsfor other values ranged from 1.0 to 5.3.

ConditionsAb-GO

ArsphenaminePeroxidase(Mg/ml)(I0^g/ml)(50^g/ml)0

+0+0

++0.07++0.7++1.4+ -+10++35++70+ +Cytotoxicity(%)«

inTNP-HEp-201447*50'63»80"100"100"100"

" Standard errors less than 10% for all values.bp < 0.001 when compared with control (nutrient medium alone).

demonstrating the specificity of the response to a-CEA-GO.Some nonspecific binding of n-GS-GO to HT-29 cells mustoccur, however, since higher concentrations of arsphenamine (10 //g/ml) gave significant killing (33 to 59%) thatwas, however, significantly less (p < 0.01) than witha-CEA-GO (66 to 90%). The contention that n-GS-GOdoes, in fact, bind to HT-29 cells but to a lesser degree thana-CEA-GO is supported by studies with a similar system ofAb-GO, lactoperoxidase, and radiolabeled iodide (24). Thenonspecific binding may in part be a reflection of aggregation in the n-GS-GO conjugate. Further purification ofconjugates and the use of specifically purified anti-CEAantibody should be helpful in increasing the selectivity ofcell kill.

The addition of partially purified CEA did result insignificant^ < 0.001) suppression of a-CEA-GO-mediatedcytotoxicity. At the lower concentration of a-CEA-GO (100¿ig/ml), cell killing was reduced by 78 to 100% whenarsphenamine (1 to 10 ¿¿g/ml)and HRP were added. At thehigher concentration of a-CEA-GO (200 ng/m\) in thecomplete system, suppression of cytotoxicity was less (34 to55%) but still significant (p < 0.001). The specificity of thisblocking activity was suggested by the fact that the sameconcentration of CEA failed to inhibit the cell killing seenwith n-GS-GO (100 or 200 ng/m\) and arsphenamine at 10Mg/ml.

In this colonie cell model, there was no significantcytocidal effect if either conjugate or cofactors were eliminated. This clearly demonstrates the importance of thecomplete system in attaining such marked cell killing andsuggests that HT-29 cells may be somewhat more resistantto nonspecific toxicity of the arsphenamine-peroxidasecombination than the HeLa or HEp-2 cells (Table 2).However, some nonspecific toxicity may well be present forHT-29 cells and might become manifest with more sensitiveassays or longer culture periods. We have in fact observed

that this is true for HT-29 cells in a similar system using thisa-CEA-GO conjugate at 200 ¿ig/mland other cofactors(lactoperoxidase and iodide) (24).

DISCUSSION

This study demonstrates another potent antibody-enzymeconjugate system that can produce affinity cytotoxicity intumor cells. In both of the antibody-tumor cell models used,selective cell killing was accomplished with Ab-GO treatment followed by culture in peroxidase and arsphenamine.Although there was some nonspecific toxicity with arsphenamine at the higher concentrations, cell killing wasinvariably increased in the presence of specifically boundAb-GO. It has long been known from studies with syphilisand trypanosomiasis that arsphenamine is considerablymore toxic when it is oxidized (2, 25). Indeed it was thisproperty that led to its selection by Ehrlich for trial in thetreatment of syphilis. Previous studies have establishedtolerable doses of arsphenamine in animals and humans,and these data will be helpful if later testing of thetumoricidal capabilities of this system is carried out in vivo.

The marked potency of this enzyme-cofactor systemmakes it a potentially promising one. Complete cell killing(100%) was obtained at relatively low levels of arsphenamine (10 /ig/m') and anti-TNP-GO conjugate (10 to 20Mg/ml). The potency of this system compares very favorablyto others which we have previously studied in the TNP cellmodel. At comparable levels of anti-TNP antibody, complement-dependent cytotoxicity was considerably less ( <43%),and selective killing with an anti-TNP-diphtheria toxinconjugate somewhat lower (78 to 86%) (19). With the sameanti-TNP-GO conjugate used for this study and differentcofactors (lactoperoxidase and iodide), only 33 to 79%

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Selective Cytotoxicity with Antibody Conjugates

Table 5Selective cytotoxicily of human colon adenocarcinoma cells with anti-CEA antibody conjugate,

arsphenamine, and peróxidos«Washed HT-29 cells (1.5 x 10s) were incubated for 30 min with PBS or with glucose oxidase

conjugates to anti-CEA (IgG) or normal goat IgG in the presence or absence of CEA (270 Mg/ml)(see "Materials and Methods"); then washed with PBS and nutrient medium; put onto microtest

plates (640 cells/well); and medium with or without arsphenamine and HRP was added. After 20 hrattached viable cells were scored by the microcytotoxicity assay. Average number of cells countedper well was determined from 6 to 12 wells.

Conditions Results

Conjugate(Mg/ml)100

or200100100100too200200200200CEA000+++00+0+0+0+Arsphen

amine(Mg/ml)01100110//.0111010111010HRP/.

None0505005050n-GS-GO05050505050505050Cells/well37.9±2.9°36.7

±4.231.5±3.440.8

±2.835.3±4.837.8

±2.035.9

±2.539.8±5.534.2±6.525.3±2.825.0±3.035.3

±7.328.5±3.318.2±2.115.5

±1.8Cyto

toxicity316070501033"34'725C52"59"

///. a-CEA-GO

100or20010010010010020020020020000+0+0+0+01110101110100505050505050505033.7±1.410.2±1.131.7

±3.612.7±2.139.2±4.16.8

±1.124.0±3.03.8±1.015.7±2.51173"1666"082"37'90a59*

°Standard errors.'•c-*Significantly different (p < 0.01;p < 0.05;p < 0.001) from control.

cytotoxicity resulted as compared to 100% killing seen inthis study (20).

The comparison of potency is even more striking in theanti-CEA colon carcinoma model. Similar treatment ofHT-29 cells with anti-CEA-GO conjugate followed bylactoperoxidase and iodide resulted in only 30 to 40%cytotoxicity, compared to the 75 to 90% seen in this study.Furthermore, at comparable levels of antibody no significant complement-mediated cytotoxicity has been seen inHT-29 with either the anti-CEA antisera or the immuno-globulin fractions (24).

The use of arsphenamine as a cofactor is not withoutsome potential disadvantages. In addition to nonspecifictoxicity, arsenicals may cause cancer (15, 25), and hypersen-sitivity to arsenobenzoates is well known (23). In spite ofthese potential problems, the potent cytocidal effect ofoxidized arsenicals makes it worthy of further explorationas a cofactor for the Ab-GO system.

Another important conclusion from this study is thatenzyme-conjugated anti-CEA antibody can be used tolocalize toxins in human colonie carcinoma cells and obtainsignificant cytotoxicity. Although CEA is probably the bestcharacterized human tumor-associated antigen and levels intumor tissue appear to be 20- to 100-fold higher than innormal tissue (12), there has been considerable doubt thatCEA specificity could be used in the immunological retardation of tumor cell growth. CEA is localized primarily inthe glycocalyx rather than the plasma membrane and iseasily shed into the circulation. Furthermore, purified CEAdoes not seem capable of stimulating peripheral lymphocytes that are sensitive to colonie tumors by other criteria(10). Our experiments with glucose oxidase conjugates andcofactors (either arsphenamine and HRP, or iodide andlactoperoxidase) are the 1st concrete evidence that underfavorable conditions selective toxicity can by obtained withanti-CEA antibody. Although some of the cytotoxicity was

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G. W. Philpott et al.

dependent upon anti-CEA antibody binding, as evidencedby the blocking with partially purified CEA (Table 5),antibodies to other cell surface antigens may have accounted for some of the cell killing. The feasibility of usingthe CEA system for immunologically mediated cytotoxicityis further suggested by recent binding studies with radio-iodinated IgG fractions of anti-CEA antisera (22) andspecifically purified anti-CEA antibody (H. F. Chao, S. C.Peiper, G. W. Philpott, C. W. Parker, and R. D. Aach,submitted for publication). We anticipate being able toimprove the selectivity of the antibody-conjugate binding byspecific purification of the anti-CEA antibody.

ACKNOWLEDGMENTS

The authors thank Steven Peiper. William Coleman, and Dr. RichardAach for assistance in the preparation of the CEA and the anti-CEAconjugate.

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2164 CANCER RESEARCH VOL. 34

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1974;34:2159-2164. Cancer Res   Gordon W. Philpott, Richard J. Bower, Keith L. Parker, et al.   Oxidase Conjugates, Peroxidase, and ArsphenamineAffinity Cytotoxicity of Tumor Cells with Antibody-Glucose

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