Antigen-specific drug-targeting used to manipulate an immune response in vivo (suppression/cytarabine/cytotoxicity/toxogen)

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 84, pp , October 1987 Immunology Antigen-specific drug-targeting used to manipulate an immune response 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 Park Memorial Institute (a unit of New York State Department of Health), 666 Elm Street, Buffalo, NY Communicated by Michael Potter, June 19, 1987 (received for review December 1, 1986) ABSTRACT The administration of dextran-conjugated cytosine arabinonucleoside (arac) to BALB/c mice at various times prior to but not subsequent to immunization with native dextran renders mice unresponsive to this thymic-independent antigen. These results demonstrate that the primary immune response to an antigen can be selectively and efficiently suppressed or eliminated in vivo by the delivery of a single dose of an appropriate antigen-cytotoxic drug conjugate. Evidence presented here indicates that the dextran-arac conjugate (toxogen) acts directly and selectively upon unprimed dextranspecifc antibody-forming cell precursors, presumably by binding to their receptors and subsequent internalization of the resultant receptor-toxogen complexes. The resistance of antigen-primed mice to the cytotoxic effect of the toxogen could result from the failure of dextran-primed cells to reexpress antigen-specific receptors, from an alternative processing of the toxogen, or from the inability of the antigen-primed cells to internalize a second round of receptor-ligand complexes. We also determined that B cells responding to thymic-dependent antigens were not affected by the prior exposure to a toxogen. The inability to eliminate or suppress the primary response to a thymic-dependent antigen via the administration of a cytotoxic drug-antigen conjugate distinguishes the thymicindependent set of B cells from the thymic-dependent B-cell repertoire. The difference between these two B-cell compartments could be due either to differences in the amount of ligand bound to receptors or to differences in the trafficking patterns of receptor-ligand complexes within each cell type. In this report we demonstrate that the primary immune response to dextran can be efficiently manipulated in vivo by the selective elimination of a defined population of immunocompetent cells (in this case, thymic-independent antigen-binding B cells). This has been accomplished with a minimal degree of disruption to the physiology and microenvironment of the host's immune system by targeting to the antigen-specific B-cell receptor a conjugate ofthe antigen and a cytotoxic drug. Dextran-binding antibody-forming cell precursors were removed by administering a conjugate of a cytotoxic drug and dextran prior to immunization with the unmodified immunogenic form of dextran. The use of monoclonal antibodies to deliver cytotoxic agents to various cells in vitro, primarily neoplastic cells, has received extensive study (1-4). The success of this approach depends upon the delivery of a cytotoxic agent to a determinant on the surface of the neoplastic cell and upon the subsequent internalization of the cytotoxic agent. The limited success in targeting drugs to tumors with monoclonal antitumor antibodies may be due to one or more of several problems. For example, the tumor most often consists of a heterogeneous mixture of a large number of cells, some of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact which do not express the cell-surface antigen that is the target of the drug delivery. The tumor cells are generally located outside of the circulatory system; therefore, access to the target is impaired. In addition, tumors characteristically shed their target surface antigens, and these molecules enter the circulation and compete with the tumor cells for binding to the antibody component of the cytotoxic conjugate (or immunotoxin). Perhaps the most fundamental problem is that, even if the toxogen can reach and bind to the tumor, there is no assurance that the toxic component will be internalized following surface binding and released in an active form into the cytoplasm. We have exploited a modified drug-targeting strategy to remove selected subsets of normal cells. Unlike the selective targeting of drugs and toxins with anti-tumor antibodies, the drug-targeting proposed here involves a limited number of cells, all of which express the appropriate target receptor and all of which are accessible via the vascular bed. In addition, when the antigen-drug conjugate is introduced, there is little or no circulating antibody to compete with the drug conjugate to remove it from the circulation prior to binding to the putative antigen-binding cells. Based upon in vitro studies, it was also assumed that appropriate antigen-binding B lymphocytes, whose receptors bound the drug-dextran complex, would rapidly and efficiently internalize the toxogen (5). A final requisite for the success of this drug-targeting concept depended upon the release of the cytotoxic drug within the cell in a form capable of arresting or killing the cell. Cytarabine (cytosine arabinonucleoside; arac) was selected because it was a cell-cycledependent drug that could be directly conjugated to antigens or 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. Dextran B1355S was oxidized with periodate as reported (6, 7) and allowed to couple to arac to form a Schiff base. The adduct, Dex-araC, was stabilized by reduction with sodium borohydride. The amount of arac incorporated in the dextran molecule was followed by using [3H]araC (Amersham) to label the arac preparation and by UV spectroscopy measurements at 26 nm. Dextran-cytidine conjugate (Dex-Cyd) was prepared in a similar way, and the amount of cytidine incorporated in the conjugate was determined by UV spectroscopy at 26 nm. The human immunoglobulin (higm) used to prepare the hapten-protein conjugate and subsequently the drug-hapten-protein toxogen was obtained by Na2SO4 Abbreviations: arac, cytosine arabinonucleoside; Dex, dextran in conjugates; KLH, keyhole limpet hemocyanin; PFC, plaque-forming cells; higm, human IgM. fto whom reprint requests should be addressed.

2 Immunology: Abu-hadid et al. precipitation of the immunoglobulins from the serum of a patient with an IgM myeloma. The higm was treated with the diazonium salt of4-aminophthalate (8), and the conjugate was oxidized with sodium periodate (.1 M) for 1 hr at 4C. The oxidized 4-azophthalate-hIgM (23.4 mg/6 ml) was allowed to couple with arac (12 mg/1.2 ml) for 1 hr at room temperature, and the adduct was stabilized by reduction with sodium borohydride (2 M, 25,ul) for 1 hr at 4C. It was determined that the resultant toxogen contained 212 mol of arac for each mol of hapten-protein carrier (higm-phthalate). 4-Azophthalate-tyramine-Dex-araC was prepared as a second toxogen for phthalate-specific B cells. Oxidized dextran (25 mg/3.4 ml) was mixed with tyramine (.2 M, 1 ml) for 15 min at room temperature, arac (95 mg/.95 ml) was added, and the reaction was continued 1 hr at room temperature. The adduct was stabilized by reduction with sodium borohydride (2 M,.5 ml). Following dialysis, the mixed adduct was allowed to react with the diazonium salt of 4-aminophthalate to form 4-azophthalate-tyramine-DexaraC; this conjugate contained 395 molecules of arac per molecule of toxogen. The conjugate of keyhole limpet hemocyanin (KLH) (Calbiochem) and the diazonium salt of 4-aminophthalate was prepared as described (8). Animals. BALB/c mice were obtained from West Seneca Laboratory (West Seneca, NY). BALB/c nu/nu mice were obtained from the National Institutes of Health (Bethesda, MD). Drug Treatment and Immunization. BALB/c mice (12-16 weeks old) were treated intravenously with 2 Al of a solution containing various amounts of the Dex-araC conjugates in phosphate-buffered saline (PBS; D-PBS; GIBCO). Control mice received 2,l of PBS, 2 Al of a solution containing 1,g of dextran B1355S and 1 mg of free arac in PBS, 2,l of a solution containing 1,ug of dextran B1355S in PBS, or 2,u of a solution containing 1 jig of Dex-Cyd conjugates. In most experiments, mice were immunized 4 days later with an emulsion of 1,g of dextran B1355S in 2,l of PBS and 2,l of complete Freund's adjuvant intraperitoneally. The timing of the administration of the toxogen and the antigen was varied, and the antidextran antibody responses were evaluated with a radioimmunoassay using [1251]iodotyramine-dextran B1355S or with a Jerne plaque assay using Dextran B1355S-conjugated sheep erythrocytes. For the thymic-dependent antibody responses, mice were treated with 4-azophthalate-hIgM-araC (1,ug) or 4-azophthalate-tyramine-Dex-araC (1 ug) 4 days prior to immunization with higm-4-azophthalate or KLH-4-azophthalate (1,g emulsified in complete Freund's adjuvant). Five days after immunization, the antiphthalate antibody response was evaluated by using a Jerne plaque assay. Radioimmunoassay. The mice were bled 7, 14, and 21 days after immunization, and the amount of anti-dextran antibodies was evaluated in a Farr binding assay as modified by Skom and Talmage (9), incubating 5,l of [125I]iodotyramine-dextran B1355S (1) that contained =1, cpm with 5,l of dilutions of antisera or standard for 1 hr and precipitating the immunocomplexes with 2,l of appropriately titrated goat anti-mouse immunoglobulin antisera. The standard for these RIAs was protein 14E, a mouse myeloma protein that binds dextran B1355S. Plaque-Forming Cells (PFC) Assay. The number of spleen cells secreting anti-dextran or anti-phthalate antibodies were determined in a Jerne plaque assay (11) as modified by Mishell and Dutton (12) using dextran-conjugated (7) or phthalate-conjugated (13) sheep erythrocytes as the target cell. Proc. Natl. Acad. Sci. USA 84 (1987) 7233 RESULTS Treatment of mice with a toxogen composed of a cytotoxic agent conjugated to an antigen proved to be very effective in suppressing the immune response. Mice, given the Dex-araC conjugate as a single intravenous injection prior to immunization, produced no detectable (<1 pg/ml) anti-dextran antibody when subsequently immunized with native dextran (Table 1). Control mice given dextran or dextran with free arac (up to 6, times the amount of arac delivered to the experimental group with the Dex-araC conjugate) all produced anti-dextran antibodies 1 week after immunization (287 and 244,ug/ml, respectively). The amount of antibody in the serum of control animals pretreated with free drug and dextran was not significantly different from that of mice pretreated with PBS (the average of 28 control mice treated with PBS and immunized with dextran was 246 ± 37 /ig/ml 1 week after immunization). Unimmunized and untreated BALB/c mice (12-18 weeks old) have undetectable levels of anti-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 to a local depot of antigen in situ, the dextran immune response was also evaluated at the single-cell level. The spleen cells were washed and assayed for the number of anti-dextran antibody-secreting cells in a modified Jerne plaque assay (11). The data presented in Fig. 1 show that both the number of dextran-specific PFC and the amount of anti-dextran antibodies were reduced by >95% in the mice pretreated with the toxogen. The observed number of antibody-producing cells in the treated mice approached background levels (i.e., the number of plaques found in unimmunized mice was -1 PFC per spleen). These data confirm the results of RIA, and the absence of dextran-specific plaques eliminates the possibility that the toxogen is acting as an antibody sink by tying up all of the antibody as it is released in situ. Thus, a single injection of the toxogen renders mice completely unresponsive to dextran. The unresponsiveness to dextran induced by the Dex-araC conjugate was selective, since the administration of Dex-araC prior to secondary immunization with the hapten-carrier conjugate of phthalate-klh had no effect upon the subsequent anti-phthalate antibody response (Table 2). To determine how long it took for the toxogen to establish drug-induced unresponsiveness prior to immunization, and to determine what effect immunization had upon the toxogen's ability to suppress the host, mice were given the Table 1. Effect of pretreatment of mice with the Dex-araC conjugate, dextran, or dextran and arac upon the dextranspecific immune response Anti-dextran antibodies after Treatment prior immunization,,ug/ml to immunization 7 days 14 days 21 days PBS 295 ± ± ± 18 Dex-araC2,6 <1* <1* <1* Dextran + arac 244 ± ± ± 55 Dextran 287 ± ± ± 42 Mice are injected intravenously with 2,ul of PBS, with 2,ul of a solution containing 1,g of Dex-araC2,6 (subscript number is the number of arac molecules per molecule of dextran) in PBS, with 2,il of a solution containing 1,g of dextran B1355S and 1 mg of free arac in PBS, or with 2 /il of a solution containing 1 ug of dextran B1355S in PBS 4 days prior to immunization. Values are means ± SEM; five mice were used in each group. *The minimal amount of protein 14E (or anti-dextran antibody) that can be measured in this assay is 1,ug/ml. Sera from unimmunized and untreated normal BALB/c mice (12-2 weeks old) have no measurable levels of anti-dextran antibodies in this assay (i.e., <1 /ig of anti-dextran antibody per ml of serum).

3 7234 Immunology: Abu-hadid et al. cn -i E (. c c CE cc. Mice Treated With Mice Treoted With Dex-AroC Isotonic Saline to C-) LiL._ 2.t_ 6 < C,) CO a 4 a I- FIG. 1. Evaluation of the number of anti-dextran antibodyproducing cells stimulated after treatment with the antigen-arac conjugate. The results are compared to the amount of anti-dextran antibodies present in the serum. The bars in the histogram represent the responses of individual mice that had been treated with 1 pag of Dex-araC26 (where the subscript specifies the number of arac molecules per molecule of dextran) or isotonic saline 4 days prior to immunization with dextran. toxogen at various times before and after immunization with native dextran. It was determined that, for complete suppression of the dextran response, the toxogen at 1 jig of toxogen per mouse with a minimum of 26 arac groups per dextran molecule had to be administered 4 days prior to immunization (Table 3). Significant suppression was observed when the dextran-drug conjugate was given just 1 day prior to immunization or as long as 21 days prior to immunization. Increasing the molar ratio of arac/dextran up to 6 did not have any effect upon the kinetics or degree of inhibition of the response to dextran. However, when the amount of Dex-araC conjugate administered to mice was increased from 1 pkg to 5 ttg, the interval between the administration of the toxogen and immunogen required for complete suppression was reduced from 4 days to 2 days (Table 3). Since arac is believed to act selectively on dividing cells or cells about to enter S phase (14, 15), the interval between the administration of toxogen and immunogen may be required to allow sufficient time for all of the target dextran-specific cells to be activated. Moreover, it has been suggested that the toxogen itself stimulates the cells to enter S phase (15), at which stage the drug may act. Table 2. Effect of pretreatment of mice with Dex-araC upon the phthalate-specific immune response Anti-phthalate responset Treatment* PFC per spleen PBS 9.94 x x 14 Dex-araC x x 14 *BALB/c mice had been primed with 1,ug of KLH-4-azophthalate before i.v. injection of PBS or 1,ug of Dex-araC26 4 days prior to a secondary immunization with KLH-4-azophthalate (1 jig). tdata are means ± SEM; five mice were used in each group. 2 Proc. Natl. Acad. Sci. USA 84 (1987) Table 3. Effect of pretreatment with various Dex-araC conjugates upon the dextran-specific immune response Time of treatment relative to immunization, Anti-dextran antibodies after immunization,,ug/ml Toxogen days 7 days 14 days Dex-araC ± 5 79 ± 35 Dex-araC ± 18 ND Dex-araC26-4 <1 <1 Dex-araCww ± ± 3 Dex-araC ± 7 56 ± 4 Dex-araC26* - 2 ND <1 Dex-araC26-1 ND 35 ± 9 Dex-araC ND 192 ± 66 Dex-araC ND 151 ± 34 Dex-araC ND 125 ± 33 Dex-araC ND 217 ± 8 Dex-araC ND 128 ± 18 PBS ± ± 32 NT 389 ± ± 66 NTt 233 ± ± 29 NTf 37 ± 9 22 ± 5 Mice were treated with 1 ug of toxogen (the subscript designates the number of arac molecules per dextran molecule) on the indicated days prior to or after immunization i.p. with 1 Ag of dextran B1355S in 2 pa of PBS, emulsified in an equal volume of complete Freund's adjuvant. The values are means ± SEM; five mice were used in each group. The minimal amount of protein 14E (or anti-dextran antibodies) that can be measured in this assay is 1,ug/ml. ND, not done; NT, no treatment. *Mice were treated with 5,g of toxogen 2 days prior to immunization with dextran B1355S. tmice were immunized with 1,Ag of Dex-Cyd86 in 2 Al of PBS emulsified in an equal volume of complete Freund's adjuvant. tmice were immunized with 1,ug of Dex-araC5 in 2 Al of PBS emulsified in an equal volume of complete Freund's adjuvant. Of particular interest in these studies was the finding that cells exposed to the native dextran prior to the toxogen (i.e., dextran-primed cells) were completely refractory to the suppressive effects of the toxogen. No significant suppression of the dextran response was observed when Dex-araC was administered 1, 2, 3, 4, or 5 days after immunization (Table 3). The refractoriness of these cells to the toxogen could result (i) from the failure of the antigen-primed cells to reexpress dextran-specific receptors that would be required for internalizing the subsequent receptor-toxogen complexes; (ii) from the failure of antigen-binding cells, following antigen-initiated differentiation, to internalize enough of the drug to be effective; or (iii) from an altered trafficking within the target cell of the internalized receptor-ligand complex. In the event of altered trafficking, the toxogen-receptor complexes may be processed by an alternative pathway that results in a sequestration or inactivation of the toxogen. Finally, administration of the toxogen after antigen stimulation could result in the toxogen binding to newly secreted antibody in the circulation, thereby preventing the toxogen from reaching the target cells. Studies have shown that treatment with high doses of dextran itself can tolerize mice (16), and it was possible that the chemical manipulation that was used to prepare the toxogen had produced a modified dextran that was an unusually good tolerogen. To eliminate this possibility, cytidine, a noncytotoxic analog of arac, was coupled to oxidized dextran by the same procedures as those used to prepare the toxogen. Mice were treated with this analog and then immunized with dextran. All mice treated with this analog produced approximately normal amounts of antidextran antibodies after immunization. In addition, we de-

4 Immunology: Abu-hadid et al. Table 4. Effect of pretreatment of athymic mice with Dex-araC upon the dextran-specific immune response Anti-dextran antibodies,t Treatment* ug/ml PBS 277 ± 6 Dex-araC5oo <1 *BALB/c nu/nu mice were treated with PBS or with 1 )ag of Dex-araC5 3 days prior to immunization with 1 gg of dextran B1355S in complete Freund's adjuvant. tthe amount of anti-dextran antibodies was evaluated by using a modified Farr binding assay. Values are means ± SEM; five mice were used in each group. The minimal amount of protein 14E that can be evaluated in this assay is 1,g/ml. Proc. Natl. Acad. Sci. USA 84 (1987) 7235 termined whether or not Dex-araC by itself was immunogenic. The administration of 1 tug of Dex-araC in Freund's complete adjuvant produced only very low amounts of anti-dextran antibodies (Table 3). Mice immunized with 1 ug of Dex-Cyd in Freund's complete adjuvant produced 233 ± 15,g of anti-dextran antibodies per ml of serum, which is similar to the level of anti-dextran antibodies observed in mice given 1,ug of native dextran in Freund's complete adjuvant-i.e., 246 ± 32 Lg/ml (Table 3). We concluded from these studies that the chemical modification of dextran per se was not responsible for the toxogen-induced unresponsiveness. Since the response to the a-1,3-linkages of dextran is largely independent of T-cell cooperation, and since it is believed from in vitro studies that helper T-cells do not bind and internalize nominal antigens, an underlying assumption was that the toxogen was binding directly to dextran-specific B lymphocytes that were the progenitors of the anti-dextran antibody-producing cells. Studies with athymic (nude) mice (Table 4) are consistent with this assumption. The nude mice that were treated with Dex-araC 3 days prior to immunization failed to respond to immunization with dextran, while the level of anti-dextran antibodies in nude mice that were not treated with the toxogen (i.e., 277,g/ml of serum) was similar to the level seen in normal BALB/c mice immunized with dextran. Since the in vivo targeting of an antigen-drug conjugate was very effective in specifically eliminating the antibody response to a thymic-independent antigen, an analogous protocol was used to determine whether or not this antigen-drug conjugate targeting would be equally effective in eliminating B cells responding to a thymic-dependent antigen. Mice were treated with either of two different antigendrug conjugates (i.e., 4-azophthalate-hIgM-araC or 4- azophthalate-tyramine-dex-arac), and the anti-phthalate antibody response was evaluated by a Jerne plaque assay after immunization with either of two different immunogenic forms of the thymic-dependent hapten-carrier complexes, 4-azophthalate-hIgM or 4-azophthalate-KLH. The results in Table 5 show that the administration of toxogen (antigenarac conjugates) prior to immunization did not produce any significant suppression in the subsequent anti-phthalate response. In fact, prior exposure to one of these toxogens (i.e., higm-phthalate-arac) actually enhanced the phthalate-specific response. Although the primary response to phthalate is typically low, the plaques reported are all inhibited by free phthalate and they are up to 6 times higher than background. Similar attempts to suppress the secondary response to phthalate with the toxogens (where the number of PFCs per spleen reaches up to 15,) were also unsuccessful. DISCUSSION Mice treated with the toxogen Dex-araC fail to respond to a subsequent stimulation with the highly immunogenic native dextran B1355S. The suppression is a function of the cytotoxicity of arac and not the result of chemical modification of dextran per se because the homologous reagent Dex-Cyd, which is structurally similar to arac but is not cytotoxic, had no suppressive effect on the anti-dextran antibody response and was found by itself to be an effective immunogen. It is suggested that the loss of the dextran response results from the cytotoxic effect of the drug because mice that have been treated with toxogen 21 days prior to immunization also fail to respond to the antigen. Since administration of [1251]iodotyramine-dextran showed that removal of the iodotyramine group could be detected within 12 hr, most of the toxogen would have been taken up by the cells in a few days to a week, and the toxogen would have been detoxified as the drug was separated from the antigen. Thus, the toxogen cannot be acting to block temporarily the antigen-binding cells because it is unlikely that such an effect would last 3 weeks, and the toxogen would not remain viable for that period of time. Moreover, we have observed that the suppressive effects of the 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 rendered immunologically tolerant for extended periods of time by exposure to an antigen while the animals were temporarily severely immunosuppressed by radiation or by high levels of immunosuppressive drugs (17). These levels are 6, times the amount of arac that we administer on our toxogen (18). These very high doses of immunosuppressive drugs can cause extensive damage to major sections of the immune system, but in the studies reported here, restricted specific targeting was expected to have minimal effects on the lymphatic microenvironment. In the earlier studies, the effects were maximal on T-dependent antibody responses, and there was the suggestion that the immunosuppressive agents had compromised T cells. In our studies we are apparently targeting directly to the B cells that specifically bind the antigen-drug conjugate. Table 5. Effect of pretreatment of BALB/c mice with 4-azophthalate-carrier-araC conjugates upon the phthalate-specific immune response Treatment prior to Anti-phthalate responses Experiment primary immunization* Immunogen PFC per spleen 1 PBS hlgm-4-azophthalate 212 ± 68 higm-phthalate-arac higm-4-azophthalate 292 ± 27 2 PBS KLH-4-azophthalate 4339 ± 246 higm-phthalate-arac KLH-4-azophthalate 226 ± 167 PBS KLH-4-azophthalate 431 ± Dex-phthalate KLH-4-azophthalate 467 ± 162 Dex-phthalate-araC KLH-4-azophthalate 421 ± 191 *Treatment occurred 4 days prior to immunization i.p. with 1 jg of immunogen in complete Freund's adjuvant. PBS indicates i.v. injection of.2 ml of PBS; 1 Ag of conjugates was administered. tvalues are means ± SEM.

5 7236 Immunology: Abu-hadid et al. The B cells responding to a thymic-independent antigen are distinguished from B cells that respond to thymic-dependent antigens, since the latter cells were not affected by the specific antigen-drug toxogen. The basis of this difference could be due to several factors. Based on the pharmacological studies of the use of arac to treat leukemia (14), it is assumed that, to eliminate the antigen-responsive B cells, the toxogen must gain access to the cell, presumably via receptor-mediated endocytosis. Subsequently, the drug must be cleaved from the internalized toxogen, as we have observed to occur with iodotyramine-dextran. Antigenic stimulation provided by the toxogen itself initiates cell proliferation, and the free arac that has been converted to the triphosphate derivative becomes incorporated into the DNA of the proliferating cells. Since much of the recent work on B-cell antigen presentation suggests that thymic-dependent antigens by themselves are not capable of initiating B-cell proliferation and are only partially degraded, we predicted that targeting of thymic-dependent antigen-cytotoxic drug conjugates would not be suppressive. Consistent with this prediction, we have examined the effect of arac conjugates on two thymic-dependent antigens, higm-4-azophthalate or KLH-4-azophthalate and bovine gamma globulin-azophenyl phosphocholine. In each case there was no observed diminution in the antibody response levels as measured by PFC assay and ELISA, respectively. The data in TableS show that prior treatment with either higm-phthalate-arac or Dexphthalate-araC toxogens had no effect on the subsequent anti-phthalate antibody response. In examining the thymicdependent anti-phosphocholine antibody response (data not shown), we found no effect on either the primary or secondary antibody response. The failure of toxogens to effect responses to thymic-dependent antigens may also be due to differences in the intracellular trafficking of the toxogen within the B cell that results in little or no release of the drug from the toxogen. The ability to deliver cytotoxic agents selectively to cell populations in vivo has many obvious potential applications in designing new approaches both to cancer therapy and to the manipulation of the immune response for addressing questions related to T- and B-cell cooperation, to receptor-ligand trafficking, to kinetics and thresholds of lymphocyte activation, and to immune regulation. With very few exceptions (19-21), the feasibility of these immunospecific targeting applications has been demonstrated only by in vitro studies. Preliminary evidence from our laboratory (22) indicates that the in vivo targeting and selective elimination of cells is not limited to antigen-specific B cells. For example, idiotype-binding cells (i.e., putative autoantiidiotype regulatory cells) have been eliminated by the in vivo administration of a single injection of an idiotype-arac conjugate. The selective depletion of the idiotype-binding cells demonstrated that these cells are crucial to the regulation of the reference idiotype (22). The use of toxogens to eliminate selected populations of cells from an immunocompetent individual represents a viable and as yet largely untapped experimental Proc. Natl. Acad. Sci. USA 84 (1987) design with which to manipulate the immune response and to evaluate the physiological significance of any cell that displays on its surface a unique and recognizable receptor or membrane-associated macromolecule that selectively binds a ligand. We wish to thank Drs. N. Klinman, A. Nisonoff, and R. Lynch for helpful discussion and suggestions concerning these studies. 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