Proc. Natl. Acad. Sci. USAVol. 84, pp. 7232-7236, October 1987Immunology

Antigen-specific drug-targeting used to manipulate an immuneresponse in vivo

(suppression/cytarabine/cytotoxicity/toxogen)

M. M. ABU-HADID*, R. B. BANKERTt, AND G. L. MAYERStt*Department of Microbiology, State University of New York at Buffalo, Buffalo, NY 14214; and tDepartment of Molecular Immunology, Roswell ParkMemorial Institute (a unit of New York State Department of Health), 666 Elm Street, Buffalo, NY 14263

Communicated by Michael Potter, June 19, 1987 (received for review December 10, 1986)

ABSTRACT The administration of dextran-conjugatedcytosine arabinonucleoside (araC) to BALB/c mice at varioustimes prior to but not subsequent to immunization with nativedextran renders mice unresponsive to this thymic-independentantigen. These results demonstrate that the primary immuneresponse to an antigen can be selectively and efficientlysuppressed or eliminated in vivo by the delivery of a single doseof an appropriate antigen-cytotoxic drug conjugate. Evidencepresented here indicates that the dextran-araC conjugate(toxogen) acts directly and selectively upon unprimed dextran-specifc antibody-forming cell precursors, presumably by bind-ing to their receptors and subsequent internalization of theresultant receptor-toxogen complexes. The resistance of anti-gen-primed mice to the cytotoxic effect of the toxogen couldresult from the failure of dextran-primed cells to reexpressantigen-specific receptors, from an alternative processing ofthe toxogen, or from the inability of the antigen-primed cells tointernalize a second round of receptor-ligand complexes. Wealso determined that B cells responding to thymic-dependentantigens were not affected by the prior exposure to a toxogen.The inability to eliminate or suppress the primary response toa thymic-dependent antigen via the administration of acytotoxic drug-antigen conjugate distinguishes the thymic-independent set of B cells from the thymic-dependent B-cellrepertoire. The difference between these two B-cell compart-ments could be due either to differences in the amount of ligandbound to receptors or to differences in the trafficking patternsof receptor-ligand complexes within each cell type.

In this report we demonstrate that the primary immuneresponse to dextran can be efficiently manipulated in vivo bythe selective elimination of a defined population of im-munocompetent cells (in this case, thymic-independent an-tigen-binding B cells). This has been accomplished with aminimal degree of disruption to the physiology and micro-environment of the host's immune system by targeting to theantigen-specific B-cell receptor a conjugate ofthe antigen anda cytotoxic drug. Dextran-binding antibody-forming cellprecursors were removed by administering a conjugate of acytotoxic drug and dextran prior to immunization with theunmodified immunogenic form of dextran.The use of monoclonal antibodies to deliver cytotoxic

agents to various cells in vitro, primarily neoplastic cells, hasreceived extensive study (1-4). The success of this approachdepends upon the delivery of a cytotoxic agent to a deter-minant on the surface of the neoplastic cell and upon thesubsequent internalization of the cytotoxic agent. The limitedsuccess in targeting drugs to tumors with monoclonal anti-tumor antibodies may be due to one or more of severalproblems. For example, the tumor most often consists of aheterogeneous mixture of a large number of cells, some of

which do not express the cell-surface antigen that is the targetof the drug delivery. The tumor cells are generally locatedoutside of the circulatory system; therefore, access to thetarget is impaired. In addition, tumors characteristically shedtheir target surface antigens, and these molecules enter thecirculation and compete with the tumor cells for binding tothe antibody component of the cytotoxic conjugate (orimmunotoxin). Perhaps the most fundamental problem isthat, even if the toxogen can reach and bind to the tumor,there is no assurance that the toxic component will beinternalized following surface binding and released in anactive form into the cytoplasm. We have exploited a modifieddrug-targeting strategy to remove selected subsets of normalcells. Unlike the selective targeting of drugs and toxins withanti-tumor antibodies, the drug-targeting proposed here in-volves a limited number of cells, all of which express theappropriate target receptor and all ofwhich are accessible viathe vascular bed. In addition, when the antigen-drug conju-gate is introduced, there is little or no circulating antibody tocompete with the drug conjugate to remove it from thecirculation prior to binding to the putative antigen-bindingcells. Based upon in vitro studies, it was also assumed thatappropriate antigen-binding B lymphocytes, whose receptorsbound the drug-dextran complex, would rapidly and effi-ciently internalize the toxogen (5). A final requisite for thesuccess of this drug-targeting concept depended upon therelease of the cytotoxic drug within the cell in a form capableof arresting or killing the cell. Cytarabine (cytosine arabino-nucleoside; araC) was selected because it was a cell-cycle-dependent drug that could be directly conjugated to antigensor antibodies without loss of the drug's cytotoxic activity (6).

EXPERIMENTAL PROCEDURES

Reagents. Dextran B1355S was a gift from M. E. Slodki(Northern Regional Research Laboratory, Peoria, IL).Cytarabine (araC) was obtained from Upjohn. DextranB1355S was oxidized with periodate as reported (6, 7) andallowed to couple to araC to form a Schiff base. The adduct,Dex-araC, was stabilized by reduction with sodium borohy-dride. The amount of araC incorporated in the dextranmolecule was followed by using [3H]araC (Amersham) tolabel the araC preparation and by UV spectroscopy mea-surements at 260 nm. Dextran-cytidine conjugate (Dex-Cyd)was prepared in a similar way, and the amount of cytidineincorporated in the conjugate was determined by UV spec-troscopy at 260 nm. The human immunoglobulin (hIgM) usedto prepare the hapten-protein conjugate and subsequentlythe drug-hapten-protein toxogen was obtained by Na2SO4

Abbreviations: araC, cytosine arabinonucleoside; Dex, dextran inconjugates; KLH, keyhole limpet hemocyanin; PFC, plaque-formingcells; hIgM, human IgM.fTo whom reprint requests should be addressed.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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precipitation of the immunoglobulins from the serum of apatient with an IgM myeloma. The hIgM was treated with thediazonium salt of4-aminophthalate (8), and the conjugate wasoxidized with sodium periodate (0.001 M) for 1 hr at 40C. Theoxidized 4-azophthalate-hIgM (23.4 mg/6 ml) was allowed tocouple with araC (120 mg/1.2 ml) for 1 hr at room temper-ature, and the adduct was stabilized by reduction withsodium borohydride (2 M, 250 ,ul) for 1 hr at 40C. It was

determined that the resultant toxogen contained 212 mol ofaraC for each mol of hapten-protein carrier (hIgM-phthal-ate). 4-Azophthalate-tyramine-Dex-araC was prepared as a

second toxogen for phthalate-specific B cells. Oxidizeddextran (25 mg/3.4 ml) was mixed with tyramine (0.2 M, 1 ml)for 15 min at room temperature, araC (95 mg/0.95 ml) was

added, and the reaction was continued 1 hr at room temper-ature. The adduct was stabilized by reduction with sodiumborohydride (2 M, 0.5 ml). Following dialysis, the mixedadduct was allowed to react with the diazonium salt of4-aminophthalate to form 4-azophthalate-tyramine-Dex-araC; this conjugate contained 3095 molecules of araC permolecule of toxogen. The conjugate of keyhole limpethemocyanin (KLH) (Calbiochem) and the diazonium salt of4-aminophthalate was prepared as described (8).

Animals. BALB/c mice were obtained from West SenecaLaboratory (West Seneca, NY). BALB/c nu/nu mice wereobtained from the National Institutes of Health (Bethesda,MD).Drug Treatment and Immunization. BALB/c mice (12-16

weeks old) were treated intravenously with 200 Al of a

solution containing various amounts of the Dex-araC conju-gates in phosphate-buffered saline (PBS; D-PBS; GIBCO).Control mice received 200 ,l of PBS, 200 Al of a solutioncontaining 100 ,g of dextran B1355S and 1 mg of free araCin PBS, 200 ,l of a solution containing 100 ,ug of dextranB1355S in PBS, or 200 ,u of a solution containing 100 jig ofDex-Cyd conjugates. In most experiments, mice were im-munized 4 days later with an emulsion of 100 ,g of dextranB1355S in 200 ,l of PBS and 200 ,l of complete Freund'sadjuvant intraperitoneally. The timing of the administrationof the toxogen and the antigen was varied, and the anti-dextran antibody responses were evaluated with a radio-immunoassay using [1251]iodotyramine-dextran B1355S orwith a Jerne plaque assay using Dextran B1355S-conjugatedsheep erythrocytes. For the thymic-dependent antibodyresponses, mice were treated with 4-azophthalate-hIgM-araC(100 ,ug) or 4-azophthalate-tyramine-Dex-araC (100 ug) 4days prior to immunization with hIgM-4-azophthalate or

KLH-4-azophthalate (100 ,g emulsified in completeFreund's adjuvant). Five days after immunization, the anti-phthalate antibody response was evaluated by using a Jerneplaque assay.Radioimmunoassay. The mice were bled 7, 14, and 21 days

after immunization, and the amount of anti-dextran antibod-ies was evaluated in a Farr binding assay as modified bySkom and Talmage (9), incubating 50 ,l of [125I]iodotyra-mine-dextran B1355S (10) that contained =100,000 cpm with50 ,l of dilutions of antisera or standard for 1 hr andprecipitating the immunocomplexes with 200 ,l of appropri-ately titrated goat anti-mouse immunoglobulin antisera. Thestandard for these RIAs was protein 104E, a mouse myelomaprotein that binds dextran B1355S.

Plaque-Forming Cells (PFC) Assay. The number of spleencells secreting anti-dextran or anti-phthalate antibodies weredetermined in a Jerne plaque assay (11) as modified byMishell and Dutton (12) using dextran-conjugated (7) or

phthalate-conjugated (13) sheep erythrocytes as the targetcell.

RESULTS

Treatment of mice with a toxogen composed of a cytotoxicagent conjugated to an antigen proved to be very effective insuppressing the immune response. Mice, given the Dex-araCconjugate as a single intravenous injection prior to immuni-zation, produced no detectable (<1 pg/ml) anti-dextranantibody when subsequently immunized with native dextran(Table 1). Control mice given dextran or dextran with freearaC (up to 6,000 times the amount of araC delivered to theexperimental group with the Dex-araC conjugate) all pro-duced anti-dextran antibodies 1 week after immunization (287and 244 ,ug/ml, respectively). The amount of antibody in theserum of control animals pretreated with free drug anddextran was not significantly different from that of micepretreated with PBS (the average of 28 control mice treatedwith PBS and immunized with dextran was 246 ± 37 /ig/ml1 week after immunization). Unimmunized and untreatedBALB/c mice (12-18 weeks old) have undetectable levels ofanti-dextran antibodies in our RIA (<1 ,ug/ml).To confirm the RIA results and to eliminate the possibility

that the toxogen was simply acting as an antibody sink due toa local depot of antigen in situ, the dextran immune responsewas also evaluated at the single-cell level. The spleen cellswere washed and assayed for the number of anti-dextranantibody-secreting cells in a modified Jerne plaque assay (11).The data presented in Fig. 1 show that both the number ofdextran-specific PFC and the amount of anti-dextran anti-bodies were reduced by >95% in the mice pretreated with thetoxogen. The observed number of antibody-producing cellsin the treated mice approached background levels (i.e., thenumber of plaques found in unimmunized mice was -1000PFC per spleen). These data confirm the results of RIA, andthe absence of dextran-specific plaques eliminates the pos-sibility that the toxogen is acting as an antibody sink by tyingup all of the antibody as it is released in situ. Thus, a singleinjection of the toxogen renders mice completely unrespon-sive to dextran. The unresponsiveness to dextran induced bythe Dex-araC conjugate was selective, since the administra-tion of Dex-araC prior to secondary immunization with thehapten-carrier conjugate of phthalate-KLH had no effectupon the subsequent anti-phthalate antibody response (Table2).To determine how long it took for the toxogen to establish

drug-induced unresponsiveness prior to immunization, andto determine what effect immunization had upon thetoxogen's ability to suppress the host, mice were given the

Table 1. Effect of pretreatment of mice with the Dex-araCconjugate, dextran, or dextran and araC upon the dextran-specific immune response

Anti-dextran antibodies after

Treatment prior immunization, ,ug/mlto immunization 7 days 14 days 21 daysPBS 295 ± 53 218 ± 44 212 ± 18Dex-araC2,6 <1* <1* <1*Dextran + araC 244 ± 50 217 ± 47 184 ± 55Dextran 287 ± 59 195 ± 52 157 ± 42

Mice are injected intravenously with 200 ,ul of PBS, with 200 ,ul ofa solution containing 100 ,g of Dex-araC2,6 (subscript number is thenumber of araC molecules per molecule of dextran) in PBS, with 200,il of a solution containing 100 ,g of dextran B1355S and 1 mg of freearaC in PBS, or with 200 /il of a solution containing 100 ug of dextranB1355S in PBS 4 days prior to immunization. Values are means ±SEM; five mice were used in each group.*The minimal amount of protein 104E (or anti-dextran antibody) thatcan be measured in this assay is 1 ,ug/ml. Sera from unimmunizedand untreated normal BALB/c mice (12-20 weeks old) have nomeasurable levels of anti-dextran antibodies in this assay (i.e., <1/ig of anti-dextran antibody per ml of serum).

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FIG. 1. Evaluation of the number of anti-dextran antibody-producing cells stimulated after treatment with the antigen-araCconjugate. The results are compared to the amount of anti-dextranantibodies present in the serum. The bars in the histogram representthe responses of individual mice that had been treated with 100 pAg ofDex-araC260 (where the subscript specifies the number of araCmolecules per molecule of dextran) or isotonic saline 4 days prior toimmunization with dextran.

toxogen at various times before and after immunization withnative dextran. It was determined that, for complete sup-pression of the dextran response, the toxogen at 100 jig oftoxogen per mouse with a minimum of 260 araC groups perdextran molecule had to be administered 4 days prior toimmunization (Table 3). Significant suppression was ob-served when the dextran-drug conjugate was given just 1 dayprior to immunization or as long as 21 days prior to immu-nization. Increasing the molar ratio of araC/dextran up to6000 did not have any effect upon the kinetics or degree ofinhibition of the response to dextran. However, when theamount of Dex-araC conjugate administered to mice wasincreased from 100 pkg to 500 ttg, the interval between theadministration of the toxogen and immunogen required forcomplete suppression was reduced from 4 days to 2 days(Table 3). Since araC is believed to act selectively on dividingcells or cells about to enter S phase (14, 15), the intervalbetween the administration of toxogen and immunogen maybe required to allow sufficient time for all of the targetdextran-specific cells to be activated. Moreover, it has beensuggested that the toxogen itself stimulates the cells to enterS phase (15), at which stage the drug may act.

Table 2. Effect of pretreatment of mice with Dex-araC upon thephthalate-specific immune response

Anti-phthalate responset

Treatment* PFC per spleen

PBS 9.094 x 104 2.77 x 104Dex-araC260 9.872 x 104 0.72 x 104

*BALB/c mice had been primed with 100 ,ug of KLH-4-azophthalatebefore i.v. injection of PBS or 100 ,ug of Dex-araC260 4 days priorto a secondary immunization with KLH-4-azophthalate (100 jig).

tData are means ± SEM; five mice were used in each group.

Table 3. Effect of pretreatment with various Dex-araCconjugates upon the dextran-specific immune response

Time of treatment Anti-dextranrelative to antibodies after

immunization, immunization, ,ug/mlToxogen days 7 days 14 days

Dex-araC330 -21 50 ± 5 79 ± 35Dex-araC330 -10 54 ± 18 NDDex-araC260 - 4 <1 <1Dex-araCww - 2 81 ± 11 33 ± 3Dex-araC260 - 2 55 ± 7 56 ± 4Dex-araC260* - 2 ND <1Dex-araC260 - 1 ND 35 ± 9Dex-araC260 + 1 ND 192 ± 66Dex-araC260 + 2 ND 151 ± 34Dex-araC260 + 3 ND 125 ± 33Dex-araC260 + 4 ND 217 ± 80Dex-araC260 + 5 ND 128 ± 18PBS - 4 327 ± 69 216 ± 32NT 389 ± 40 363 ± 66NTt 233 ± 15 161 ± 29NTf 37 ± 9 22 ± 5

Mice were treated with 100 ug of toxogen (the subscript designatesthe number of araC molecules per dextran molecule) on the indicateddays prior to or after immunization i.p. with 100 Ag of dextranB1355S in 200 pA of PBS, emulsified in an equal volume of completeFreund's adjuvant. The values are means ± SEM; five mice wereused in each group. The minimal amount of protein 104E (oranti-dextran antibodies) that can be measured in this assay is 1,ug/ml. ND, not done; NT, no treatment.*Mice were treated with 500 ,g of toxogen 2 days prior to immuni-zation with dextran B1355S.tMice were immunized with 100 ,Ag of Dex-Cyd860 in 200 Al of PBSemulsified in an equal volume of complete Freund's adjuvant.tMice were immunized with 100 ,ug of Dex-araC500 in 200 Al of PBSemulsified in an equal volume of complete Freund's adjuvant.

Of particular interest in these studies was the finding thatcells exposed to the native dextran prior to the toxogen (i.e.,dextran-primed cells) were completely refractory to thesuppressive effects of the toxogen. No significant suppres-sion of the dextran response was observed when Dex-araCwas administered 1, 2, 3, 4, or 5 days after immunization(Table 3). The refractoriness of these cells to the toxogencould result (i) from the failure of the antigen-primed cells toreexpress dextran-specific receptors that would be requiredfor internalizing the subsequent receptor-toxogen complex-es; (ii) from the failure of antigen-binding cells, followingantigen-initiated differentiation, to internalize enough of thedrug to be effective; or (iii) from an altered trafficking withinthe target cell of the internalized receptor-ligand complex. Inthe event of altered trafficking, the toxogen-receptor com-plexes may be processed by an alternative pathway thatresults in a sequestration or inactivation of the toxogen.Finally, administration of the toxogen after antigen stimula-tion could result in the toxogen binding to newly secretedantibody in the circulation, thereby preventing the toxogenfrom reaching the target cells.

Studies have shown that treatment with high doses ofdextran itself can tolerize mice (16), and it was possible thatthe chemical manipulation that was used to prepare thetoxogen had produced a modified dextran that was anunusually good tolerogen. To eliminate this possibility,cytidine, a noncytotoxic analog of araC, was coupled tooxidized dextran by the same procedures as those used toprepare the toxogen. Mice were treated with this analog andthen immunized with dextran. All mice treated with thisanalog produced approximately normal amounts of anti-dextran antibodies after immunization. In addition, we de-

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Table 4. Effect of pretreatment of athymic mice with Dex-araCupon the dextran-specific immune response

Anti-dextranantibodies,t

Treatment* ug/mlPBS 277 ± 60Dex-araC5oo <1

*BALB/c nu/nu mice were treated with PBS or with 100 )ag ofDex-araC500 3 days prior to immunization with 100 gg of dextranB1355S in complete Freund's adjuvant.tThe amount of anti-dextran antibodies was evaluated by using amodified Farr binding assay. Values are means ± SEM; five micewere used in each group. The minimal amount of protein 104E thatcan be evaluated in this assay is 1 ,g/ml.

termined whether or not Dex-araC by itself was immunogen-ic. The administration of 100 tug of Dex-araC in Freund'scomplete adjuvant produced only very low amounts ofanti-dextran antibodies (Table 3). Mice immunized with 100ug of Dex-Cyd in Freund's complete adjuvant produced 233± 15 ,g of anti-dextran antibodies per ml of serum, which issimilar to the level of anti-dextran antibodies observed inmice given 100 ,ug of native dextran in Freund's completeadjuvant-i.e., 246 ± 32 Lg/ml (Table 3). We concluded fromthese studies that the chemical modification of dextran per sewas not responsible for the toxogen-induced unresponsive-ness.

Since the response to the a-1,3-linkages of dextran islargely independent of T-cell cooperation, and since it isbelieved from in vitro studies that helper T-cells do not bindand internalize nominal antigens, an underlying assumptionwas that the toxogen was binding directly to dextran-specificB lymphocytes that were the progenitors of the anti-dextranantibody-producing cells. Studies with athymic (nude) mice(Table 4) are consistent with this assumption. The nude micethat were treated with Dex-araC 3 days prior to immunizationfailed to respond to immunization with dextran, while thelevel of anti-dextran antibodies in nude mice that were nottreated with the toxogen (i.e., 277 ,g/ml of serum) wassimilar to the level seen in normal BALB/c mice immunizedwith dextran.

Since the in vivo targeting of an antigen-drug conjugatewas very effective in specifically eliminating the antibodyresponse to a thymic-independent antigen, an analogousprotocol was used to determine whether or not this anti-gen-drug conjugate targeting would be equally effective ineliminating B cells responding to a thymic-dependent anti-gen. Mice were treated with either of two different antigen-drug conjugates (i.e., 4-azophthalate-hIgM-araC or 4-azophthalate-tyramine-Dex-araC), and the anti-phthalate an-tibody response was evaluated by a Jerne plaque assay afterimmunization with either oftwo different immunogenic formsof the thymic-dependent hapten-carrier complexes, 4-azo-

phthalate-hIgM or 4-azophthalate-KLH. The results in Table5 show that the administration of toxogen (antigen-araC conjugates) prior to immunization did not produce anysignificant suppression in the subsequent anti-phthalate re-sponse. In fact, prior exposure to one of these toxogens (i.e.,hIgM-phthalate-araC) actually enhanced the phthalate-spe-cific response. Although the primary response to phthalate istypically low, the plaques reported are all inhibited by freephthalate and they are up to 60 times higher than background.Similar attempts to suppress the secondary response tophthalate with the toxogens (where the number of PFCs perspleen reaches up to 150,000) were also unsuccessful.

DISCUSSIONMice treated with the toxogen Dex-araC fail to respond to a

subsequent stimulation with the highly immunogenic nativedextran B1355S. The suppression is a function of the cyto-toxicity of araC and not the result of chemical modificationof dextran per se because the homologous reagent Dex-Cyd,which is structurally similar to araC but is not cytotoxic, hadno suppressive effect on the anti-dextran antibody responseand was found by itself to be an effective immunogen. It issuggested that the loss of the dextran response results fromthe cytotoxic effect of the drug because mice that have beentreated with toxogen 21 days prior to immunization also failto respond to the antigen. Since administration of [1251]iodo-tyramine-dextran showed that removal of the iodotyraminegroup could be detected within 12 hr, most of the toxogenwould have been taken up by the cells in a few days to a week,and the toxogen would have been detoxified as the drug wasseparated from the antigen. Thus, the toxogen cannot beacting to block temporarily the antigen-binding cells becauseit is unlikely that such an effect would last 3 weeks, and thetoxogen would not remain viable for that period of time.Moreover, we have observed that the suppressive effects ofthe Dex-araC toxogen persist for at least 5 months.The experiments reported here are quite different from

several classical studies in which adult animals were renderedimmunologically tolerant for extended periods of time byexposure to an antigen while the animals were temporarilyseverely immunosuppressed by radiation or by high levels ofimmunosuppressive drugs (17). These levels are 600,000times the amount of araC that we administer on our toxogen(18). These very high doses of immunosuppressive drugs cancause extensive damage to major sections of the immunesystem, but in the studies reported here, restricted specifictargeting was expected to have minimal effects on thelymphatic microenvironment. In the earlier studies, theeffects were maximal on T-dependent antibody responses,and there was the suggestion that the immunosuppressiveagents had compromised T cells. In our studies we are

apparently targeting directly to the B cells that specificallybind the antigen-drug conjugate.

Table 5. Effect of pretreatment of BALB/c mice with 4-azophthalate-carrier-araC conjugatesupon the phthalate-specific immune response

Treatment prior to Anti-phthalate responsesExperiment primary immunization* Immunogen PFC per spleen

1 PBS hlgM-4-azophthalate 212 ± 68hIgM-phthalate-araC hIgM-4-azophthalate 2920 ± 2070

2 PBS KLH-4-azophthalate 4339 ± 2460hIgM-phthalate-araC KLH-4-azophthalate 2260 ± 1670PBS KLH-4-azophthalate 431 ± 166

3 Dex-phthalate KLH-4-azophthalate 467 ± 162Dex-phthalate-araC KLH-4-azophthalate 421 ± 191

*Treatment occurred 4 days prior to immunization i.p. with 100 jg of immunogen in complete Freund'sadjuvant. PBS indicates i.v. injection of 0.2 ml of PBS; 100 Ag of conjugates was administered.tValues are means ± SEM.

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The B cells responding to a thymic-independent antigen aredistinguished from B cells that respond to thymic-dependentantigens, since the latter cells were not affected by thespecific antigen-drug toxogen. The basis of this differencecould be due to several factors. Based on the pharmacolog-ical studies of the use of araC to treat leukemia (14), it isassumed that, to eliminate the antigen-responsive B cells, thetoxogen must gain access to the cell, presumably via recep-tor-mediated endocytosis. Subsequently, the drug must becleaved from the internalized toxogen, as we have observedto occur with iodotyramine-dextran. Antigenic stimulationprovided by the toxogen itself initiates cell proliferation, andthe free araC that has been converted to the triphosphatederivative becomes incorporated into the DNA of the pro-liferating cells. Since much of the recent work on B-cellantigen presentation suggests that thymic-dependent anti-gens by themselves are not capable of initiating B-cellproliferation and are only partially degraded, we predictedthat targeting of thymic-dependent antigen-cytotoxic drugconjugates would not be suppressive. Consistent with thisprediction, we have examined the effect of araC conjugateson two thymic-dependent antigens, hIgM-4-azophthalate orKLH-4-azophthalate and bovine gamma globulin-azophenylphosphocholine. In each case there was no observed dimi-nution in the antibody response levels as measured by PFCassay and ELISA, respectively. The data in TableS show thatprior treatment with either hIgM-phthalate-araC or Dex-phthalate-araC toxogens had no effect on the subsequentanti-phthalate antibody response. In examining the thymic-dependent anti-phosphocholine antibody response (data notshown), we found no effect on either the primary or second-ary antibody response. The failure of toxogens to effectresponses to thymic-dependent antigens may also be due todifferences in the intracellular trafficking of the toxogenwithin the B cell that results in little or no release of the drugfrom the toxogen.The ability to deliver cytotoxic agents selectively to cell

populations in vivo has many obvious potential applicationsin designing new approaches both to cancer therapy and tothe manipulation of the immune response for addressingquestions related to T- and B-cell cooperation, to recep-tor-ligand trafficking, to kinetics and thresholds of lympho-cyte activation, and to immune regulation. With very fewexceptions (19-21), the feasibility of these immunospecifictargeting applications has been demonstrated only by in vitrostudies. Preliminary evidence from our laboratory (22) indi-cates that the in vivo targeting and selective elimination ofcells is not limited to antigen-specific B cells. For example,idiotype-binding cells (i.e., putative autoantiidiotype regula-tory cells) have been eliminated by the in vivo administrationof a single injection of an idiotype-araC conjugate. Theselective depletion ofthe idiotype-binding cells demonstratedthat these cells are crucial to the regulation of the referenceidiotype (22). The use of toxogens to eliminate selectedpopulations of cells from an immunocompetent individualrepresents a viable and as yet largely untapped experimental

design with which to manipulate the immune response and toevaluate the physiological significance of any cell that dis-plays on its surface a unique and recognizable receptor ormembrane-associated macromolecule that selectively binds aligand.

We wish to thank Drs. N. Klinman, A. Nisonoff, and R. Lynch forhelpful discussion and suggestions concerning these studies. Wethank Jenni Loyall for performing the plaque assays and Art Trottand Richard Maturski for technical assistance. M.M.A. is a recipientof a Fulbright Scholarship through America-Mideast Educational &Training Services, Inc. A major portion of the data is taken from thedissertation to be submitted by M.M.A. to the State University ofNew York at Buffalo in partial fulfillment of the requirements for thedegree of Doctor of Philosophy. This work was supported byNational Institutes of Health Grants CA25253, CA33462, andCA22786.

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11. Jerne, N. K., Nordin, A. A. & Henry, C. (1963) in Cell BoundAntibodies, eds. Amos, B. & Koprowski, H. (Wistar InstitutePress, Philadelphia), pp. 109-125.

12. Mishell, R. I. & Dutton, R. W. (1967) J. Exp. Med. 126,423-442.

13. Bankert, R. B., Mazzaferro, D. & Mayers, G. L. (1981) Hy-bridoma 1, 47-58.

14. Pallavicini, M. G. (1984) Pharmacol. Ther. 25, 207-238.15. Valeriote, F. (1982) Med. Pediatr. Oncol. Suppl. 1, 5-26.16. Howard, J. G. & Courtenay, B. M. (1975) Immunology 29,

599-610.17. Weigle, W. 0. (1976) Adv. Imrmnunol. 16, 61-122.18. Gordon, R. O., Wade, M. E. & Mitchell, M. S. (1969) J.

Immunol. 103, 233-243.19. Colly, L. P., van Bekkam, D. W. & Hagenbeek, A. (1984)

Leukemia Res. 8, 953-963.20. Diener, E., Diner, U. E., Sinha, A., Xie, S. & Vergidis, R.

(1986) Science 231, 148-150.21. Mayers, G. L., Bankert, R. B. & Abu-hadid, M. M. (1986)

Prog. Immunol. 6th Int. Congr. Immunol., 487.22. Abu-hadid, M. M., Bankert, R. B. & Mayers, G. L. (1986)

Prog. Immunol. 6th Int. Congr. Immunol., 495.

Proc. Natl. Acad. Sci. USA 84 (1987)

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