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Intern. Rev. Immunol. 7 , 1991, pp. 289-302 Reprints available directly from the publisher Photocopying permitted by license only a1991 Harwood Academic Publishers GmbH Printed in the United States of America
Anti-Idiotype Monoclonal Antibodies as Vaccines for Human Cancer MALAYA BHATTACHARYA-CHATTERJEE,? KENNETH A. FOON,? and HEINZ KOHLER* ?Division of Clinical Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA gIDEC Pharmaceuticals Corporation, 11099 N. Torrey Pines Road, La Jolla, California 92037, USA (Received March 13, 1991; accepted April 10, 1991)
The anti-idiotype therapy approach has been tested and has shown to be effective in several animal models including the L1210lGZL tumor system in DBM2 mice. Very recently, anti-idiotype antibodies (Ab2) have also been used in human trials. In this review, the generation and characterization of Ab2s which can be used as potential vaccine candidates for two human tumor systems-leukemia4ymphona and gastrointestinal carcinoma have been discussed. We have generated syngeneic monoclonal idiotypic cascades for two different human tumorassociated antigens (TAA) gp37 and carcinoembryonic antigen (CEA). In both cascades we have produced TAA mimicking monoclonal Ab2s and monoclonal anti-anti-idiotypes (Ab3) which bind to the original TAA. Modulation of immune responses in cancer patients by Ab2 immunization will be an important consideration in future studies. KEYWORDS: idiorype immunization, CEA, human leukemiallymphoma
INTRODUCTION Cancer patients are often immunosuppressed and also tolerant to some tumor-associated antigens (TAA). Triggering an active immune response to such TAA represents an important challenge in cancer therapy. One approach uses tumor derived material as the immunogen. As an alternative to the use of tumor antigens or tumor cells, the network hypothesis of Niels Jerne [l] offers an elegant approach to vaccine therapy using the socalled internal image antigens [2-61. According to the network concept, immunization with a given antigen will generate the production of antibodies against this antigen termed Abl. This Abl can generate a series of anti-idiotype (Id) antibodies against Abl termed Ab2. Some of these Ab2 molecules can effectively mimic the three dimensional structures of external antigens. These particular anti-idiotypes called Ab2P [2-61, which fit into the paratopes of Abl, can induce specific immune responses similar to responses induced by nominal antigen. Anti-idiotypic antibodies of the p type express the internal image of the antigen recognized by the Abl antibody and can be used as surrogate antigens. Immunization with Ab2P can lead to the generation of anti-anti-idiotype antibodies (Ab3) that recognize the corresponding original antigen identified by the Abl. Because of this Abllike reactivity, the Ab3 is also called Abl’ to indicate that it might differ in its other idiotopes from Abl. This cyclic nature of complementary binding sites and idiotopes is the basis for 289
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the approach to idiotope vaccines. Several such Ab2P have been used in animal models to trigger the immune system to induce specific and protective immunity against bacterial [7, 81, viral [9, 101 (including HIV) [ll] and parasitic infections [12]. The administration of Ab2P as surrogate tumor-associated antigens represents another potential application of the idiotype vaccine concept. The anti-idiotype hybridoma route of making surrogate antigens could alleviate the problem of preparing large amounts of purified antigens associated with a given tumor. Also, the internal antigens which are expressed in a different molecular environment may be able to overcome the immunosuppression and break the tolerance in the host by stimulating “silent” clones, or by activating T cell help, making the overall immune response stronger than the nominal antigen is capable to do [13,14]. In addition, by virtue of being proteins, anti-idiotype antibodies can be easily manipulated and coupled to potential immunogenic carrier molecules to make them T cell dependent antigens which can receive full T help. Furthermore, idiotype-based tumor antigens used for immunotherapy are free of the potential danger of transmitting tumor viruses. This alternative and safe approach to active immunotherapy with the use of internal image antigens as vaccines has generated a great deal of interest in recent years in the treatment of cancer and infectious diseases [reviewed in 151. In this report, we will briefly summarize our data on the use of internal image antiidiotypic antibodies in an experimental tumor system and then discuss our efforts towards the generation of anti-idiotype vaccines for human tumors.
THE MURINE TUMOR SYSTEM The experimental tumor used in our ongoing tumor idiotype vaccine studies is designated L1210/GZL and was derived from DBN2 mice and selected as a drug-resistant mutant. L1210/GZL expresses a surface protein which cross-reacts with the gp52 envelope of mouse mammary tumor virus (MMTV) [16]. This gp52 protein serves in our model as the tumorassociated antigen (TAA). Among a number of monoclonal antibodies (mAb) reacting with gp52 protein, llCl has the highest affinity and was used as an Abl to generate anti-idiotypic mAbs in non-syngeneic and syngeneic hosts [171. Interestingly, all of these monoclonal anti-idiotypes could inhibit the binding of the llCl (Abl) to the gp52 tumor antigen. Immunizing DBA/2 with monoclonal anti-idiotypess induced immune responses related to the TAA on the L1210/GZLtumor. The responses induced by immunization with 2F10 or 3A4, two antigen-site specific anti-idiotypes, produced anti-TAA titers and increased the llCl idiotype (Abl) expression [17, 181. In addition to humoral responses, the two antiidiotypes stimulated DTH and cytotoxic T cells specific for the L1210/GZL tumor [19]. Finally, we demonstrated the induction of T helper cells which recognize the 2F10 and 3A4 idiotypes [ 181. Collectively, these data suggest that the 2F10 and 3A4 anti-idiotypes are internal image [5] or network antigens [20]. Interestingly, only immunization with 2F10 provided protection against tumor growth [21, 221 demonstrating that only 2F10 is a “network” antigen. The term “network antigen” was coined for the biologically effective Ab2 [20]. According to this definition, network antigens may or may not carry the immunochemical criteria of internal images but must be able to induce specific and biologically beneficial immune responses in a given system or disease. The key to understanding network antigens can be found by dissecting the regulating network operating in host defense against disease. The disease can be cancer, autoimmunity or infection. This led us to a series of experiments designed to dissect the regulatory network which appears to control anti-idiotype induced tumor immunity. These studies were also extended
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to an analysis of idiotope-related changes at the T helper and serum levels induced by the growing tumor. We have recently completed a study on the molecular basis of a tumor antigen induced idiotypic cascade. In this study [22], we compared the structural, idiotypic and network properties of the protective and nonprotective anti-idiotypic antibodies. In summary, the biologic activity to protect could be traced back to unique structures on the V, regions and idiotypic mimicry of the TAA was evident at the linear sequence homology between the gp52 TAA and the 2F10 V, sequence. In addition, the CDR2 region also had homology with a T cell epitope on gp52. The biologic basis of effective idiotypic mimicry was studied at the level of Ab3 induced by the Ab2. Idiotype inhibition analysis using Ab3 induced by either protective or nonprotective Ab2, revealed differences. Thus, there was evidence for differences among the Abl-Ab2-Ab3 cascade induced by protective and nonprotectiveantiidiotype. However, to demonstrate conclusively that sequence homology has biologic meaning, one must demonstrate peptide-induced tumor immunity similar to what has been shown in the reovirus system [23, 241. We will continue our studies on the murine idiotype vaccine model to address some of these unresolved issues; however, we feel strongly that similar analysis and comparison of biological and structural characteristics of anti-idiotypes which mimic a human tumorassociated antigen are warranted at this time. The results in animals give confidence that a similar cascade could be induced in patients with monoclonal Ab2ps.
ANTI-IDIOTYPE ANTIBODIES AND HUMAN CANCER Limited experience in human trials using anti-idiotype to stimulate immunity against tumors has shown promising results [25, 261. Herlyn et al. have generated polyclonal goat Ab2 against C017-1A, a murine mAb to human gastrointestinal cancer cells. Advanced colorectal cancer patients immunized with alum-precipitated goat Ab2 anti-C017-1A developed Ab3 with Abl-like activity [25]. The Ab3 had binding specificities on the surfaces of cultured tumor cells similar to the specificity of Abl. The therapeutic efficacy of such a protocol however, remains to be determined. Ferrone and his colleagues, in a recent study demonstrated that intradermal injection of anti-idiotype mAb MK2-23, which mimics a high molecular weight human melanoma antigen, elicited anti-tumor antibody response in the host. Furthermore, reduction in the size of metastatic lesions were observed in a minority of the immunized patients [26]. Recently we have generated monoclonal idiotypic cascades for two different human tumor-associated antigens. The first cascade originated from a T cell leukemiallymphoma [27, 281 and the other from carcinoembryonic antigen (CEA) [29]. In both cascades we have produced TAA mimicking monoclonal anti-idiotypes and monoclonal anti-antiidiotypes (Ab3) which bind to the original TAA.
HUMAN T CELL LEUKEMIA TAA IDIOTYPIC CASCADE To explore the feasibility of the anti-idiotype vaccine approach for human cancer, we initiated our studies with human T cell leukemias and lymphomas. T cell acute lymphoblastic leukemia (T-ALL), represents approximately 20% of the cases of ALL and more than half of these patients are not cured with chemotherapy. CutaneousT cell lymphoma (CTCL) is an indolent but incurable disease. Exploitation of new approaches to treating T-ALL and
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CTCL are important. Our approach is to use anti-idiotype antibodies as antigen surrogates for the induction of therapeutic immunity. A number of murine mAbs against various unique human T-cell leukemia-associated antigens have been described [30]. The mAb SN2 was selected for production of anti-idiotypesbecause it defines the protein moiety of a relatively unique human T-cell leukemidlymphoma-associatedcell-surface glycoprotein, gp37 (MW 37,000), which has been partially characterized [31]. The gp37 antigen is expressed by the majority of T-ALL and CTCL cells and has a limited heterogeneity among cells removed from patients at different stages of therapy or phases of the cell cycle. SN2 has a high specificity for T-leukemidlymphoma cells and does not react with any normal tissues including thymocytes, lymphocytes, bone marrow cells, liver, kidney, etc. A weak reactivity with platelets was initially detected using a radioimmunoassay (RIA); however, this reactivity could not be confirmed by fluorescencetechniques (K. Foon, unpublished data). SN2 was used to generate a number of syngeneic anti-idiotype antibodies and two were further characterized as internal image anti-idiotypes [27]. 4EA2 (IgG1, K) and 4DC6 (IgG1, A) induce TAA specific antibodies in mice and rabbits. These polyclonal Ab3s react with gp37 and MOLT-4 cells and thus has the same specificity as SN2. We proceeded then to obtain monoclonal Ab3s from anti-idiotype immunized mice [28]. The immunoreactivity of murine Ab3 and Abl was compared against a panel of human leukernidlymphoma cell lines and normal EBV virus transformed B cell lines by cell binding RIA. The binding specificity of monoclonal Ab3 was identical with SN2. We also compared the immunoreactivity of monoclonal Ab3 and Abl (SN2) against many normal human adult tissues by a sensitive immunoperoxidase (IP) staining method [32]. The IP assay was performed on 5pm sections of frozen tissue blocks with Ab2 used as control antibody. None of the normal adult tissues tested showed any significant staining with monoclonal Abl (SN2), or Ab3 indicating the absence of gp37 in those organs. Frozen sections of MOLT-4 showed intense staining with both Abl and Ab3. We tested CTCL cells from 6 patients and 5 out of 6 were strongly positive for gp37. Since the gp37 antigen has been partially purified and characterized [31], it was of interest to determine whether the Ab3 antibodies obtained from different species would precipitate the same glycoprotein as Abl. For this, the iodinated gp37 antigen was immunoprecipitated by different Ab3 preparations as well as Abl and analyzed by SDSPAGE. The results indicate that both murine monoclonal Ab3 lA3 and 2C3 precipitated a 37K major band which had the same mobility as the band precipitated by SN2 (Abl). Similar results were obtained with rabbit polyclonal Ab3 [28]. Therefore, these Ab3 antibodies can be designated as Abl’ antibodies because they have the same target antigen as SN2. Thus, in these studies we have generated a monoclonal idiotypic cascade specific for a human TAA (Figure 1). These TAA-specific humoral responses are induced in the mouse which is the origin of the Abl and in a different species (rabbit). By this criteria, such anti-idiotypic antibodies are referred to as internal images or Ab2P [4, 61. Collectively, these data indicate that these Ab2P may be useful as an antigen substitute for the generation of anti-tumor immunity in human T cell leukernidlymphoma patients. A Phase I clinical trial will be performed using the Ab2P 4DC6.
CARCINOEMBRYONIC ANTIGEN (CEA) IDIOTYPIC CASCADE CEA is one of the first TAA which has been characterized and extensively used as a tumor specific marker in cancer patients [33]. CEA is expressed by a broad spectrum of human
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FIGURE 1 Idiotype network for human T cell leukemidlymphoma.
tumor cells and can serve as a target for immunological intervention. CEA is also shed into the circulation and thus must be taken into consideration in antibody-based therapeutic approaches. Successful radioimaging of in vivo tumors with anti-CEA mAbs has been obtained in the presence of circulating CEA without clinical toxicity [34, 351. A number of anti-CEA mAbs have been generated from different laboratories [36-401. Some of the antibodies react with an epitope which is specific for the glycoprotein whereas others react with common antigenic determinants which are also present on CEA related antigens such as normal cross-reacting antigen (NCA), normal glycoprotein (NGP), biliary glycoprotein, etc. These latter antigens are commonly found on normal human tissues. Among the most specific antibodies is the one described by Koprowski et al. [36] and Mitchell [37]. This antibody designated 116NS-3d or 8019 recognizes a high molecular weight CEA primarily associated with human colonic and pancreatic carcinoma. The 8019 epitope is unique to CEA and not present on other members of the antigen family. There is virtually no cross-reactivity with normal adult tissues including granulocytes. The restricted specificity of this mAb together with its high binding capacity to a representative colon carcinoma cell line LS174T makes it an excellent target for generating Ab2 hybridomas. Furthermore, extensive sequence data on the 180kD CEA which is recognized by 8019, are available [41]. This allows a search for sequence homologies between Ab2 and CEA. This antibody has been used as an Abl to generate monoclonal anti-idiotype (Ab2) in syngeneic BALB/c mice. Several Ab2s were produced which react specifically with 8019 and not with isotype matched immunoglobulins. While the original 8019 Abl was an IgM, the particular clone we have obtained is an IgG1, suggesting that a spontaneous switch mutant was cloned. At least two Ab2s were obtained which reacted with paratope-related idiotypes on 8019 (Abl). One of these Ab2, 3H1 (IgG1,K) appears to functionally mimic CEA. The binding of labeled Abl to Ab2 was inhibited by purified CEA, and we also demonstrated that 3H1 could induce an anti-CEA antibody response in syngeneic BALB/c mice. This was accomplished by immunization of syngeneic mice with KLH-coupled 3H1 in the presence of complete and incomplete Freunds adjuvant. Syngeneic mice immunized with 100 p.g of 3HI-KLH produced measurable Ab3 responses after three immunizations.
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Next, the sera from 3H1-immunized mice were tested for antibody binding to CEA. A purified CEA (Rougier Biotech, Montreal, Canada) was used to reduce the risk of obtaining false positive results due to nonspecific binding. As shown in Figure 2, immunization with 3H1 induced antibodies that bound to insolubilized CEA. All immunized mice (six in two groups) developed anti-CEA antibody measured by an ELISA assay. Control sera from preimmune mice or mice immunized with an unrelated Ab2-KLH (4EA2) did not show binding to CEA. In parallel experiments, the binding of the same antisera was compared on a plate coated with unrelated ovarian tumor glycoprotein. The maximum binding obtained in each case was between 0.3 to 0.4 OD unit which was the same obtained with the phosphate buffered saline-bovine serum albumin (PBS-BSA) control. To determine the reactivity with cell-surface CEA, cultured human colon carcinoma LS174T cells were tested by immune flow cytometry. As shown in Figure 3, sera from 3H1immunized mice showed distinct binding (B) that was similar to the binding pattern obtained with 8019 (Abl) (A). No significant binding was obtained with human B cell lymphoma cells which do not express CEA (Fig. 3D). Next the question whether these Ab3 share 8019 idiotopes was addressed. Sera from syngeneic mice immunized with 3H1, at 1/10 dilution, inhibited binding of iodinated 3H1 (Ab2) to Abl by 90%. No inhibitionby preimmune sera or sera from mice immunized with the unrelated Ab2, 4EA2-KLH was observed. Although steric hindrance by Ab3 binding
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FIGURE 2 Binding of polyclonal mice Ab3 sera to purified CEA by ELISA. The plate was coated with pure CEA (50 ng/well) and reacted with Ab3 sera (1/100 dilution) from mice immunized with Ab2 (3Ht) and unrelated Ab2 (4EA2) of the same isotype. F’reinmune sera and PBS-BSA were also used as controls. The results are presented as the mean OD at 405 nm 5 SEM; n = 6 for all groups. In a parallel control experiment, the same sera were checked on a plate coated with unrelated ovarian tumor glycoprotein. There was no reactivity, the OD value obtained was comparable to that obtained with PBS-BSA control. (Reproduced from the Journal of Immunology [29])
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Fluorescence FIGURE 3 Flow microfluorimetry analysis of LS174-Tcells with mice Ab3 sera. n m o r cells were reacted with Abl (A) and Ab3 sera (1/50 dilution) from mice immunized with Ab2, 3H1 (B). The reaction was developed with goat anti-mouse F(ab’)2 IgG-FITC labeled antibody. Re-immune mice sera were used as control (C). In D, human B cell lymphoma cells that do not express CEA were reacted with mice Ab3 sera raised against 3H1. Symbols used (-) Abl or Ab3 or preimmune sera, (. . .) PBS-BSA. (Reproduced from the Journal of Zmmunofogy [29]).
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cannot be excluded in these assays, the results suggest the presence of Ab3 antibodies that share idiotopes with Abl (8019). If the Ab2, 3H1 is a true internal image, then it should induce the production of Agspecific Ab3 in the absence of exposure to Ag in a genetically unrestricted way and across species barriers. To confirm this, we immunized rabbits with Ab2 3H1 for the production of Ab3 that might share idiotopes with Abl and exhibit identical binding specificity. The antisera from rabbits 729 and 730, immunized with 3H1, at 1/10dilution, inhibited binding of iodinated 8019 to Ab2 by 88 and 57% respectively. No significant inhibition was obtained with preimmune rabbit sera. It has been previously shown that mAb 8019 specifically immunoprecipitatesthe 180,OOO m.w. CEA by SDS-PAGE analysis [37]. To confirm that the Ab3 induced by 3H1 was specific for the CEA molecule, semipurified extract of CEA was separated by SDS-PAGE and transblotted to nitrocellulose filters. One filter strip (Fig. 4, lane 2) was stained with Buffalo black. There were two overlapping bands at the 180,000 m.w. region (CEA) and one major band at the 50,000 m.w. region (normal cross-reacting Ag) and a few minor low
FIGURE 4 Western blotting analysis of CEA extract with Abl and Ab3. Semipurified CEA extract was separated by SDS-PAGE and transblotted into nitrocellulose filters. Lane 1 (m.w. marker) and lane 2 (CEA ext) were stained with Buffalo black. The rest of the lanes were incubated with antibody as follows: lane 3 (8019 IgG1, 10 mg/ml), lane 4 (rabbit Ab3 sera against 3H1,1/500 dilution in PBS-BSA-Tween 20), lane 5 (preimmune rabbit sera, 11500 dilution in PBS-BSA-Tbeen 20) and lane 6 (control rabbit Ab3 sera against 4EA2, 1/500 dilution in PBS-BSATbeen 20). The reaction was developed by ELISA assay using goat anti-mouse immunoglobulin and goat antirabbit immunoglobulin reagents conjugated with alkaline phosphatase. (Reproduced from the Journal of Immunology ~ 9 1 ) .
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m.w. bands. The remaining filter strips were then incubated with mAb 8019, rabbit Ab3 sera, and control rabbit sera immunized with unrelated Ab2P 4EA2 (isotype matched). The reaction was developed by the ELISA assay. mAb 8019 (Fig. 4, lane 3) and rabbit Ab3 (lane 4) immunoprecipitated only molecules with a molecular mass of 180,000 Da from this complex mixture. The materials that were not precipitated by mAb 8019 or rabbit Ab3 sera, contained a wide range of lower m.w. CEA-related antigen. There was no reactivity with preimmune (Fig. 4, lane 5 ) or control sera (lane 6). The Western blotting analysis confirmed the specificity of mAb 8019 and the reactivity of rabbit Ab3 with 180,000 m.w. CEA. We have recently generated anti-anti-Id mAb from syngeneic mice immunized with 3H1 [29]. mAb Ab3 (culture supernatantdiluted 1/5) also immunoprecipitatedthe same 180,000 m.w. CEA as Abl by Western blotting analysis (Fig. 5). The idiotype-anti-idiotype interactions in the 8019 (Abl) system have been depicted in Figure 6. We compared the reactivities of Abl 8019 with that of Ab3 (polyclonal as well as monoclonal) by a sensitive immunoperoxidase assay on normal colon and colonic tumor specimens surgically removed from patients. The pattern of reactivity of Ab3 on both
FIGURE 5 Western blotting analysis of CEA with Abl and monoclonal Ab3 culture supernatant. Experimental details are same as in Figure 4. Lane 1 (8019 IgGl, 10 mglml), lane 2 (mAb culture supernatant, 1/5 dilution in PBS-BSA-Ween 20), lane 3 (control myeloma P3-653 cult supernatant). (Reproduced from the Journal of Immunology 1291).
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FTGURE 6 Idiotype network for human gastrointestinal carcinoma
normal and malignant colonic tissues was almost identical to that obtained with Abl. There was no reaction with normal colonic mucosa. mAb 8019 and polyclonal mouse and rabbit Ab3 sera, as well as Ab3 mAb showed intense reactivity with colonic tumors. The staining was apical in gland-like structures and granular (cytoplasmic) in less differentiated areas. There were, however, minor, subtle differences between the staining patterns obtained with mAb Abl (IgG1, K ) and mAb Ab3 (IgM, K). Reaction with 8019 (Abl) resulted in the staining of tumor cells as well as secreted mucinous materials whereas reaction with mAb Ab3 resulted in the staining of tumor cells with no staining of secreted mucin [Fig. 71. Viale et al. [42] also observed minor differences in the staining patterns of Abl and Ab3 mAbs and hypothesized that these differences might reside in the nature of the immunogens used to raise the two mAbs (nominal antigen vs internal image). We plan to compare Abl and Ab3 mAb on a large panel of normal and malignant tissues. Collectively, these data suggest that such Ab2P may be useful as an antigen substitute for the induction of therapeutic immunity in CEA positive tumor patients.
DISCUSSION The anti-idiotype therapy approach has been tested and has shown to be effective in several animal tumor models [15, 43-45]. Particularly interesting are the observations that some but not all Ab2ps can induce protective immunity against tumor growth. Structure/function relationships would have predictive values in active immunotherapy using TAA-mimicking anti-Id. There exists very few anti-idiotype reagents to human cancer that have been characterizedserologically and biologically that show promise for clinical trials. To the best of our knowledge, 3H1 is the only potential anti-mAb reagent available for anti-idiotype immunization therapy of CEA positive GI cancer patients. The primary questions to be resolved are whether this anti-idiotypereagent can evoke an Ab3 as well as cellular immune response in patients; whether any Ab3 so derived behaves as an Abl-like antibody and whether this Ab3 and/or cellular immunity can mediate a potential antitumor effect. There
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FIGURE 7 Immunoperoxidase staining of colonic adenocarcinomas by monoclonal Abl and monoclonal Ab3. Serial sections were stained with a, 8019 IgGl (10 mg/ml, note intense staining in tumor area (T) and mucinous materials(M); b, Ab3 culture supernatant (neat), intense staining in the tumoral area (T)but no staining in the mucinous materials (M); c,P3-653 myeloma supernatant (neat), no reactivity in tumoral area (T) or mucinous materials (M). All sections were counterstained with hematoxylin X 240. (Reproduced from the Journal of immunology [29]).
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is evidence that certain Ab2 can induce an immune response in “silent clones” of lymphocytes which are not activated by mere exposure to nominal antigen [13, 141. Active immunization of patients with Ab2P might generate an Ab3 (Abl’) response which will hopefully be able to recruit human effector cells or complement to destroy the tumor by antibody-dependent-cell-mediatedcytotoxicity (ADCC) or complement-mediated cytotoxicity (CMC). In addition, anti-idiotype antibodies could induce various T cell activities such as stimulation of DTH and cytotoxic T cells specific for the tumor. Syngeneic monoclonal anti-idiotype antibodies have been successfully utilized as specific immunogens to elicit both T and B cell mediated immunity in our experimental L1210/GZL tumor system [19] and viral systems [lo, 461. In order to break tolerance to TAA, patients might have to be immunized with Ab2P after conjugation with a potential immunogenic carrier and mixed with an appropriate adjuvant. We expect to gather valuable information from these studies. Human therapeutic trials with anti-idiotypesare still in the very early stages. The knowledge gained from our attempts to modulate immune response of cancer patients with anti-idiotype reagents (3H1 for GI cancer and 4DC6 for T cell Ieukemiallymphoma)will help us to design similar studies with other human tumor systems. Since we have characterized a syngeneic monoclonal cascade of Abl, Ab2 and Ab3, starting with human CEA and gp37, these will enable us to analyze the structural basis of idiotypic antigen mimicry at the molecular level. This might help in the rational design of a more meaningful Hanti-idiotype vaccine in the future.
Acknowledgments This work was supported by United States Public Health Service Grants CA47860, CA51434. We thank Susan Morey and Zie Rue Zhang for excellent technical assistance and Pat Alvarado for typing of the manuscript.
References 1. Jerne, N. K., Towards anetwork theory of the immune system. Ann. Immunol. (Paris) U S C , 373-389, 1974. 2. Nisonoff, A. and Lamoyi, E., Implications of the presence of an internal image of the antigen in anti-idiotypic antibodies: possible application to vaccine production. Clin. Immunol. Immurwpurhol. 21, 397-406, 1981. 3. Kohler, H., The immune network revisited. In: Idiotypy in Biology and Medicine, edited by H. Kohler, J. Urbain, and F! Cazenave, New York: Academic Press, 1985, 3-28. 4. Kohler, H., Muller, S., and Bona, C., Internal antigen and the immune network. Proc. SOC.Exp. Biol. Med. 178, 189-193, 1985. 5 . Jerne, N. K., Roland, J., and Cazenave, A., Recurrent idiotypes and internal images. EMBO J. 1, 243-248, 1982. 6. Bona, C. A. and Kohler, H., Anti-idiotypic antibodies and internal images. In: Probesfor Recepror Structure andFunction. Vol. 4 , edited by J. C. Venter, C. M. Fraser, and J. Lindstrom, New York: Alan R. Liss, 1984, 141-150. 7. McNamara, M. K., Ward, R. E., and Kohler, H., Monoclonal idiotopes vaccine against Streptococcus pneumoniae infection. Science 226, 1325-1326, 1984. 8. Stein, K. E. and Soderstrom, T. Neonatal administration of anti-idiotype primes for protection against Escherichia coli K13 infection in mice. J. Exp. Med. 160, 1001-1011, 1984. 9. Kennedy, R. C., Eichberg, J. W., Lanford, R. E., and Dreesman, G. R., Anti-idiotypicantibody vaccine for type B viral hepatitis in chimpanzees. Science 232. 220-223, 1986. 10. Ertl, H. C. J. and Finberg, R. W., Sendai virus-specific T-cell clones: induction of cytolyticT cells by an antiidiotypic antibody directed against a helper T-cell clone. Proc. Narl. Acad. Sci. USA 81, 2850 2854, 1984. 11. Chanh, T. C., Dreesman, G. R., and Kennedy, R. C., Monocloncal antiidiotypic antibody mimics the CD4 receptor and binds human immunodeficiency virus. Proc. Narl. Acud. Sci. USA 84, 3891-3895, 1987. 12. Sacks, D. L., Esser, K. M., and Sher, A., Immunization of mice against African trypanosomiasis using antiidiotypic antibodies. J. Exp. Med. 155, 1108-1119, 1982.
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13. Bone, C., Herber-Katz, E., Paul, E. W., Idiotype-antiidiotyp regulation. I. Immunization with a levanbinding myeloma protein leads to the appearance of auto-anti-(anti-idiotype)antibodies and to the activation of silent clones. J. Enp. Med. 153, 951-967, 1981. 14. Cazenave, €? A,, Idiotypic-anti-idiotypic regulation of antibody synthesis in rabbits. Proc. Narl. Acod. Sci. USA 74, 5122-5125, 1977. 15. Bhattacharya-Chatterjee, M. and Kohler, H., Anti-idiotype tumor vaccines. In: Immunobiology ofProreins and Peprides. K Vaccines, edited by M. Z. Atassi, New York: Plenum, 1989, 113-127. 16. Rapp, L. and Fuji, H., Differential antigenic expression of DBN2 lymphoma L1210 and its sublines crossreactivity with C3H mammary tumors as defined by syngeneic monoclonal antibodies. Cancer Res. 43,25922599, 1983. 17. Raychaudhuri, S., Saeki, Y., Fuji, H., and Kohler, H., 'hmor-specific idiotype vaccines. 1. Generation and characterization of internal image tumor antigen. J. Immunol. W7, 1743-1749, 1987. 18. Raychaudhuri, S., Saeki, Y., Chen, J-J, and Kohler, H., 'hmor-specific idiotype vaccine IV Analysis of the idiotypic network in tumor immunity. J. Immunol. 139, 3902-3910, 1987. 19. Raychaudhuri, S., Saeki, Y., Chen, J-J, and Kohler, H., 'hmor-specific idiotype vaccines. 111. Induction of T helper cells by anti-idiotype and tumor cell. J. Immunol.139, 2096-2102, 1987. 20. Kohler, H., Kieber-Emmons, T., Srinivasan, S., Kaveri, S., Morrow, W. J. W., Muller, S., Kang, C., and Raychaudhuri, S., Revised immune network concepts. Clin. Immutwl. Immunoparh. 52, 104-116, 1989. 21. Raychaudhuri, S., Saeki, Y., Chen, J-J, Iribe, H., Fuji, H., and Kohler, H., 'hmor-specific idiotype vaccines. II. Analysis of the tumor-related network response induced by the tumor and by internal image antigens (AbZb). J. Immunol. 139, 271-278, 1987. 22. Raychaudhuri, S., Kang, C-Y,Kaveri, S. V, Kieber-Emmons, T., and Kohler, H., 'Thmor idiotypic vaccines VII. Analysis and correlation of structural idiotypic and biological properties of protective and non-protective Ab2s. J. Immunol. 145, 760-767, 1990. 23. Williams, W. V.,Guy, H. R., Rubin, D. H., Robey, E, Myers, J. N., Kieber-Emmons, T., Weiner, D. B., and Greene, I., Sequence of the cell attachment sites of reovirus type 3 and its anti-idiotypic antireceptor antibody: modeling of their three-dimensional structures. Proc. Natl. Acad. Sci. USA 85,6488-6495, 1988. 24. Williams, W. V, London, S. D., Weiner, D. B., Wardsworth, S., Berzofsky, J. A,, Robey, E, Rubin, D. H., and Greene, M. I., Immune response to a molecularly defined internal image idiotope. J. Immunol.142, 43924400, 1989. 25. Herlyn, D., Wettendorf, M., Schmoll, E., Hopoulos, D., Schedel, I., Dreikhausen, U., Raab, R., Ross, A. H., Jaksche, H., Scriba, M., and Koprowski, H., Anti-idiotype immunization of cancer patients: Modulation of immune response. Proc. Narl. Acad. Sci. USA 84, 8055-8059, 1987. 26. Ferrone, S. Z., Chen, J., Yang, H., Yamada, M., Zheng, Y., and Mittleman, A,, Active specific immunotherapy with murine antiidiotypic monoclonal antibodies which bear the internal image of the human high molecular weight melanoma-associated antigen (HMW-MAA). Proc. Am. Assoc. Cancer Res. 31, p. 474, 1990. 27. Bhattacharya-Chatterjee, M., Pride, M. W., Seon, B. K., Kohler, H., Idiotype vaccines against human T cell acute lymphoblastic leukemia (T-ALL). I. Generation and characterizations of biologically active monoclonal anti-idiotopes. J. Immunol. 139, 1354-1360, 1987. 28. Bhattacharya-Chatterjee, M., Chatterjee, S. K., Vasile, S., Seon, B. K., and Kohler, H., Idiotype vaccines against human T cell leukemia. 11. Generation and characterization of a monoclonal idiotype cascade (Abl, Ab2 and Ab3). J. Immunol. 141, 1398-1403, 1988. 29. Bhattacharya-Chatterjee, M., Mukerjee, S., Biddle. W., Foon, K. A,, and Kohler, H., Murine monoclonal anti-idiotype antibody as a potential network antigen for human carcinoembryonic antigen. J. Immunol. 145, 2758-2765, 1990. Tebbi, C. K., Chervinsky, D., and Fukukawa, T., Monoclonal 30. Seon, B. K., Negoro, S., Barcos, M. €?, antibody SN2 defining a human T cell leukemia-associated cell surface glycoprotein. J. Immunol. 132,20892095, 1984. 31. Seon, B. K., Fukukawa, T., Jackson, A. L., Chervinsky, D., Tebbi, C. K., Freeman, A. L., and Matsuzaki, H., Human T-cell leukemia-associated cell surface glycoprotein gp37: studies with three monoclonal antibodies and a rabbit antiserum. Mol. Immunol. 23, 569-580, 1986. 32. Gangopadhyay, A,, Bhattacharya, M., Chatterjee, S. K., Barlow, J. J., and Tsukada, Y.,Immunoperoxidase localization of a high molecular weight mucin recognized by monoclonal antibody 1D3. Cancer Res. 45, 1744-1752, 1985. 33. Gold, I! and Freedman, S. O., Demonstration of tumor-specific antigens in human colonic carcinoma by immunological tolerance and adsorption techniques. J. Exp. Med. 12, 439-462, 1965. 34. Goldenberg, D. M., Targeting of cancer with radiolabeled antibodies: prospects for imaging and therapy. Arch. Path. Lab. Med. 112, 580-587. 1988. 35. Sharkey, R. M., Goldenberg, M. H., Goldenberg, H., Lee, R. E., Ballance, C., Pawlyk, D., Varga, D., and Hansen H. J., Murine monoclonal antibodies against carcinoembryonic antigen: immunological, pharmacokinetic and targeting properties in humans. Cancer Res. 50, 2823-2831, 1990. 36. Koprowski, H., Steplewski, Z., Mitchell, K., Herlyn, M., Herlyn, D., and Fuhrer, E, Colorectal carcinoma antigens detected by hybridoma antibodies. Somatic Cell Genet. 5, 957-972, 1979.
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37. Mitchell, K. E,A carcinoembryonic antigen (CEA) specific monoclonal hybridoma antibody that reacts only with high molecular weight CEA. Cancer Immunol. Immumtkr. 10, 1-5, 1980. 38. Muraco, R., Wunderlich, D., Thor, A., Lundy, J., Noguchi, I?, Cunningham, R., and Schlom, J., Definition by monoclonal antibodies of a repertoire of epitopes on carcinoembryonicantigen differentiallyexpressed in buman colon carcinomas versus n o d adult tissues. Cancer Res. 46, 5769-5780, 1985. 39. Haggarty, A., Legler, C., Krantz, M. J., and Fuks, A., Epitopes of carcinoembryonic antigen defined by monoclonal antibodies prepared from mice immunized with purified carcinoembryonicantigen or HCT-8R cells. Cancer Res. 46, 300-309, 1986. 40. Zhang, H-Z., Ordonez, G., Batsakis, J. G., and Chan, J. C., Monoclonal antibody recognizing a carcinoembryonic antigen epitope differentially expressed in human colonic carcinoma versus normal adult colon tissues. Cancer Res. 49, 5766-5773, 1989. 41. Paxton, R.J., Mooser, G., Pande, H., Lee, T. D., and Shivley, J. E., Sequence analysis of carcinoembryonic antigen, identification of glycosilation sites and homology with the immunoglobulin supergene family. Proc. Natl. Acad. Sci. USA 84, 920-924, 1987. 42. Vide, G., Hamini, G., Grassi, E, Buffa, R., Natali, F! G., Pelagi, M., Leoni, E,Menard, S., and Siccardi, A. G., Idiotypic replica of an anti-human tumor-associated antigen monoclonal antibody: analysis of monoclonal Abl and Ab3 fine specificity. J. Immunol.143, 4338-4344, 1989. 43. Chen, J. J., Saeki, Y.,Shi, L., and Kohler, H., Synergisticanti-tumor effects with combined “internal image” anti-idiotypes and chemotherapy. J. Immunol.143, 1053-1057, 1989. 44. Campbell, M. J., Esserman, L., and Levy, R., Immunotherapy of established murine B cell lymphoma. Combination of idiotype and cyclophosphamide.J. Immunol. 141, 3227-3233, 1988. 45. Nelson, K. A,, George, E., Swenson, C., Forstrom, J. W., and Hellstrom, K. E., Immunotherapy of murine sarcomas with auto-anti-anti-idiotypicmonoclonal antibodies which bind to tumor-specific T cells. J. Immunol. 139, 2110-2117, 1987. 46. Graulton, G. N. and Greene, M. I., Anti-idiotypic antibodies of reovirus as biochemical and immunological mimics. Intern. Rev. Immun. 1, 79-90, 1986.
Intern. Rev. Immunol. 7 , 1991, pp. 289-302 Reprints available directly from the publisher Photocopying permitted by license only a1991 Harwood Academic Publishers GmbH Printed in the United States of America
Anti-Idiotype Monoclonal Antibodies as Vaccines for Human Cancer MALAYA BHATTACHARYA-CHATTERJEE,? KENNETH A. FOON,? and HEINZ KOHLER* ?Division of Clinical Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA gIDEC Pharmaceuticals Corporation, 11099 N. Torrey Pines Road, La Jolla, California 92037, USA (Received March 13, 1991; accepted April 10, 1991)
The anti-idiotype therapy approach has been tested and has shown to be effective in several animal models including the L1210lGZL tumor system in DBM2 mice. Very recently, anti-idiotype antibodies (Ab2) have also been used in human trials. In this review, the generation and characterization of Ab2s which can be used as potential vaccine candidates for two human tumor systems-leukemia4ymphona and gastrointestinal carcinoma have been discussed. We have generated syngeneic monoclonal idiotypic cascades for two different human tumorassociated antigens (TAA) gp37 and carcinoembryonic antigen (CEA). In both cascades we have produced TAA mimicking monoclonal Ab2s and monoclonal anti-anti-idiotypes (Ab3) which bind to the original TAA. Modulation of immune responses in cancer patients by Ab2 immunization will be an important consideration in future studies. KEYWORDS: idiorype immunization, CEA, human leukemiallymphoma
INTRODUCTION Cancer patients are often immunosuppressed and also tolerant to some tumor-associated antigens (TAA). Triggering an active immune response to such TAA represents an important challenge in cancer therapy. One approach uses tumor derived material as the immunogen. As an alternative to the use of tumor antigens or tumor cells, the network hypothesis of Niels Jerne [l] offers an elegant approach to vaccine therapy using the socalled internal image antigens [2-61. According to the network concept, immunization with a given antigen will generate the production of antibodies against this antigen termed Abl. This Abl can generate a series of anti-idiotype (Id) antibodies against Abl termed Ab2. Some of these Ab2 molecules can effectively mimic the three dimensional structures of external antigens. These particular anti-idiotypes called Ab2P [2-61, which fit into the paratopes of Abl, can induce specific immune responses similar to responses induced by nominal antigen. Anti-idiotypic antibodies of the p type express the internal image of the antigen recognized by the Abl antibody and can be used as surrogate antigens. Immunization with Ab2P can lead to the generation of anti-anti-idiotype antibodies (Ab3) that recognize the corresponding original antigen identified by the Abl. Because of this Abllike reactivity, the Ab3 is also called Abl’ to indicate that it might differ in its other idiotopes from Abl. This cyclic nature of complementary binding sites and idiotopes is the basis for 289
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the approach to idiotope vaccines. Several such Ab2P have been used in animal models to trigger the immune system to induce specific and protective immunity against bacterial [7, 81, viral [9, 101 (including HIV) [ll] and parasitic infections [12]. The administration of Ab2P as surrogate tumor-associated antigens represents another potential application of the idiotype vaccine concept. The anti-idiotype hybridoma route of making surrogate antigens could alleviate the problem of preparing large amounts of purified antigens associated with a given tumor. Also, the internal antigens which are expressed in a different molecular environment may be able to overcome the immunosuppression and break the tolerance in the host by stimulating “silent” clones, or by activating T cell help, making the overall immune response stronger than the nominal antigen is capable to do [13,14]. In addition, by virtue of being proteins, anti-idiotype antibodies can be easily manipulated and coupled to potential immunogenic carrier molecules to make them T cell dependent antigens which can receive full T help. Furthermore, idiotype-based tumor antigens used for immunotherapy are free of the potential danger of transmitting tumor viruses. This alternative and safe approach to active immunotherapy with the use of internal image antigens as vaccines has generated a great deal of interest in recent years in the treatment of cancer and infectious diseases [reviewed in 151. In this report, we will briefly summarize our data on the use of internal image antiidiotypic antibodies in an experimental tumor system and then discuss our efforts towards the generation of anti-idiotype vaccines for human tumors.
THE MURINE TUMOR SYSTEM The experimental tumor used in our ongoing tumor idiotype vaccine studies is designated L1210/GZL and was derived from DBN2 mice and selected as a drug-resistant mutant. L1210/GZL expresses a surface protein which cross-reacts with the gp52 envelope of mouse mammary tumor virus (MMTV) [16]. This gp52 protein serves in our model as the tumorassociated antigen (TAA). Among a number of monoclonal antibodies (mAb) reacting with gp52 protein, llCl has the highest affinity and was used as an Abl to generate anti-idiotypic mAbs in non-syngeneic and syngeneic hosts [171. Interestingly, all of these monoclonal anti-idiotypes could inhibit the binding of the llCl (Abl) to the gp52 tumor antigen. Immunizing DBA/2 with monoclonal anti-idiotypess induced immune responses related to the TAA on the L1210/GZLtumor. The responses induced by immunization with 2F10 or 3A4, two antigen-site specific anti-idiotypes, produced anti-TAA titers and increased the llCl idiotype (Abl) expression [17, 181. In addition to humoral responses, the two antiidiotypes stimulated DTH and cytotoxic T cells specific for the L1210/GZL tumor [19]. Finally, we demonstrated the induction of T helper cells which recognize the 2F10 and 3A4 idiotypes [ 181. Collectively, these data suggest that the 2F10 and 3A4 anti-idiotypes are internal image [5] or network antigens [20]. Interestingly, only immunization with 2F10 provided protection against tumor growth [21, 221 demonstrating that only 2F10 is a “network” antigen. The term “network antigen” was coined for the biologically effective Ab2 [20]. According to this definition, network antigens may or may not carry the immunochemical criteria of internal images but must be able to induce specific and biologically beneficial immune responses in a given system or disease. The key to understanding network antigens can be found by dissecting the regulating network operating in host defense against disease. The disease can be cancer, autoimmunity or infection. This led us to a series of experiments designed to dissect the regulatory network which appears to control anti-idiotype induced tumor immunity. These studies were also extended
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to an analysis of idiotope-related changes at the T helper and serum levels induced by the growing tumor. We have recently completed a study on the molecular basis of a tumor antigen induced idiotypic cascade. In this study [22], we compared the structural, idiotypic and network properties of the protective and nonprotective anti-idiotypic antibodies. In summary, the biologic activity to protect could be traced back to unique structures on the V, regions and idiotypic mimicry of the TAA was evident at the linear sequence homology between the gp52 TAA and the 2F10 V, sequence. In addition, the CDR2 region also had homology with a T cell epitope on gp52. The biologic basis of effective idiotypic mimicry was studied at the level of Ab3 induced by the Ab2. Idiotype inhibition analysis using Ab3 induced by either protective or nonprotective Ab2, revealed differences. Thus, there was evidence for differences among the Abl-Ab2-Ab3 cascade induced by protective and nonprotectiveantiidiotype. However, to demonstrate conclusively that sequence homology has biologic meaning, one must demonstrate peptide-induced tumor immunity similar to what has been shown in the reovirus system [23, 241. We will continue our studies on the murine idiotype vaccine model to address some of these unresolved issues; however, we feel strongly that similar analysis and comparison of biological and structural characteristics of anti-idiotypes which mimic a human tumorassociated antigen are warranted at this time. The results in animals give confidence that a similar cascade could be induced in patients with monoclonal Ab2ps.
ANTI-IDIOTYPE ANTIBODIES AND HUMAN CANCER Limited experience in human trials using anti-idiotype to stimulate immunity against tumors has shown promising results [25, 261. Herlyn et al. have generated polyclonal goat Ab2 against C017-1A, a murine mAb to human gastrointestinal cancer cells. Advanced colorectal cancer patients immunized with alum-precipitated goat Ab2 anti-C017-1A developed Ab3 with Abl-like activity [25]. The Ab3 had binding specificities on the surfaces of cultured tumor cells similar to the specificity of Abl. The therapeutic efficacy of such a protocol however, remains to be determined. Ferrone and his colleagues, in a recent study demonstrated that intradermal injection of anti-idiotype mAb MK2-23, which mimics a high molecular weight human melanoma antigen, elicited anti-tumor antibody response in the host. Furthermore, reduction in the size of metastatic lesions were observed in a minority of the immunized patients [26]. Recently we have generated monoclonal idiotypic cascades for two different human tumor-associated antigens. The first cascade originated from a T cell leukemiallymphoma [27, 281 and the other from carcinoembryonic antigen (CEA) [29]. In both cascades we have produced TAA mimicking monoclonal anti-idiotypes and monoclonal anti-antiidiotypes (Ab3) which bind to the original TAA.
HUMAN T CELL LEUKEMIA TAA IDIOTYPIC CASCADE To explore the feasibility of the anti-idiotype vaccine approach for human cancer, we initiated our studies with human T cell leukemias and lymphomas. T cell acute lymphoblastic leukemia (T-ALL), represents approximately 20% of the cases of ALL and more than half of these patients are not cured with chemotherapy. CutaneousT cell lymphoma (CTCL) is an indolent but incurable disease. Exploitation of new approaches to treating T-ALL and
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CTCL are important. Our approach is to use anti-idiotype antibodies as antigen surrogates for the induction of therapeutic immunity. A number of murine mAbs against various unique human T-cell leukemia-associated antigens have been described [30]. The mAb SN2 was selected for production of anti-idiotypesbecause it defines the protein moiety of a relatively unique human T-cell leukemidlymphoma-associatedcell-surface glycoprotein, gp37 (MW 37,000), which has been partially characterized [31]. The gp37 antigen is expressed by the majority of T-ALL and CTCL cells and has a limited heterogeneity among cells removed from patients at different stages of therapy or phases of the cell cycle. SN2 has a high specificity for T-leukemidlymphoma cells and does not react with any normal tissues including thymocytes, lymphocytes, bone marrow cells, liver, kidney, etc. A weak reactivity with platelets was initially detected using a radioimmunoassay (RIA); however, this reactivity could not be confirmed by fluorescencetechniques (K. Foon, unpublished data). SN2 was used to generate a number of syngeneic anti-idiotype antibodies and two were further characterized as internal image anti-idiotypes [27]. 4EA2 (IgG1, K) and 4DC6 (IgG1, A) induce TAA specific antibodies in mice and rabbits. These polyclonal Ab3s react with gp37 and MOLT-4 cells and thus has the same specificity as SN2. We proceeded then to obtain monoclonal Ab3s from anti-idiotype immunized mice [28]. The immunoreactivity of murine Ab3 and Abl was compared against a panel of human leukernidlymphoma cell lines and normal EBV virus transformed B cell lines by cell binding RIA. The binding specificity of monoclonal Ab3 was identical with SN2. We also compared the immunoreactivity of monoclonal Ab3 and Abl (SN2) against many normal human adult tissues by a sensitive immunoperoxidase (IP) staining method [32]. The IP assay was performed on 5pm sections of frozen tissue blocks with Ab2 used as control antibody. None of the normal adult tissues tested showed any significant staining with monoclonal Abl (SN2), or Ab3 indicating the absence of gp37 in those organs. Frozen sections of MOLT-4 showed intense staining with both Abl and Ab3. We tested CTCL cells from 6 patients and 5 out of 6 were strongly positive for gp37. Since the gp37 antigen has been partially purified and characterized [31], it was of interest to determine whether the Ab3 antibodies obtained from different species would precipitate the same glycoprotein as Abl. For this, the iodinated gp37 antigen was immunoprecipitated by different Ab3 preparations as well as Abl and analyzed by SDSPAGE. The results indicate that both murine monoclonal Ab3 lA3 and 2C3 precipitated a 37K major band which had the same mobility as the band precipitated by SN2 (Abl). Similar results were obtained with rabbit polyclonal Ab3 [28]. Therefore, these Ab3 antibodies can be designated as Abl’ antibodies because they have the same target antigen as SN2. Thus, in these studies we have generated a monoclonal idiotypic cascade specific for a human TAA (Figure 1). These TAA-specific humoral responses are induced in the mouse which is the origin of the Abl and in a different species (rabbit). By this criteria, such anti-idiotypic antibodies are referred to as internal images or Ab2P [4, 61. Collectively, these data indicate that these Ab2P may be useful as an antigen substitute for the generation of anti-tumor immunity in human T cell leukernidlymphoma patients. A Phase I clinical trial will be performed using the Ab2P 4DC6.
CARCINOEMBRYONIC ANTIGEN (CEA) IDIOTYPIC CASCADE CEA is one of the first TAA which has been characterized and extensively used as a tumor specific marker in cancer patients [33]. CEA is expressed by a broad spectrum of human
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FIGURE 1 Idiotype network for human T cell leukemidlymphoma.
tumor cells and can serve as a target for immunological intervention. CEA is also shed into the circulation and thus must be taken into consideration in antibody-based therapeutic approaches. Successful radioimaging of in vivo tumors with anti-CEA mAbs has been obtained in the presence of circulating CEA without clinical toxicity [34, 351. A number of anti-CEA mAbs have been generated from different laboratories [36-401. Some of the antibodies react with an epitope which is specific for the glycoprotein whereas others react with common antigenic determinants which are also present on CEA related antigens such as normal cross-reacting antigen (NCA), normal glycoprotein (NGP), biliary glycoprotein, etc. These latter antigens are commonly found on normal human tissues. Among the most specific antibodies is the one described by Koprowski et al. [36] and Mitchell [37]. This antibody designated 116NS-3d or 8019 recognizes a high molecular weight CEA primarily associated with human colonic and pancreatic carcinoma. The 8019 epitope is unique to CEA and not present on other members of the antigen family. There is virtually no cross-reactivity with normal adult tissues including granulocytes. The restricted specificity of this mAb together with its high binding capacity to a representative colon carcinoma cell line LS174T makes it an excellent target for generating Ab2 hybridomas. Furthermore, extensive sequence data on the 180kD CEA which is recognized by 8019, are available [41]. This allows a search for sequence homologies between Ab2 and CEA. This antibody has been used as an Abl to generate monoclonal anti-idiotype (Ab2) in syngeneic BALB/c mice. Several Ab2s were produced which react specifically with 8019 and not with isotype matched immunoglobulins. While the original 8019 Abl was an IgM, the particular clone we have obtained is an IgG1, suggesting that a spontaneous switch mutant was cloned. At least two Ab2s were obtained which reacted with paratope-related idiotypes on 8019 (Abl). One of these Ab2, 3H1 (IgG1,K) appears to functionally mimic CEA. The binding of labeled Abl to Ab2 was inhibited by purified CEA, and we also demonstrated that 3H1 could induce an anti-CEA antibody response in syngeneic BALB/c mice. This was accomplished by immunization of syngeneic mice with KLH-coupled 3H1 in the presence of complete and incomplete Freunds adjuvant. Syngeneic mice immunized with 100 p.g of 3HI-KLH produced measurable Ab3 responses after three immunizations.
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Next, the sera from 3H1-immunized mice were tested for antibody binding to CEA. A purified CEA (Rougier Biotech, Montreal, Canada) was used to reduce the risk of obtaining false positive results due to nonspecific binding. As shown in Figure 2, immunization with 3H1 induced antibodies that bound to insolubilized CEA. All immunized mice (six in two groups) developed anti-CEA antibody measured by an ELISA assay. Control sera from preimmune mice or mice immunized with an unrelated Ab2-KLH (4EA2) did not show binding to CEA. In parallel experiments, the binding of the same antisera was compared on a plate coated with unrelated ovarian tumor glycoprotein. The maximum binding obtained in each case was between 0.3 to 0.4 OD unit which was the same obtained with the phosphate buffered saline-bovine serum albumin (PBS-BSA) control. To determine the reactivity with cell-surface CEA, cultured human colon carcinoma LS174T cells were tested by immune flow cytometry. As shown in Figure 3, sera from 3H1immunized mice showed distinct binding (B) that was similar to the binding pattern obtained with 8019 (Abl) (A). No significant binding was obtained with human B cell lymphoma cells which do not express CEA (Fig. 3D). Next the question whether these Ab3 share 8019 idiotopes was addressed. Sera from syngeneic mice immunized with 3H1, at 1/10 dilution, inhibited binding of iodinated 3H1 (Ab2) to Abl by 90%. No inhibitionby preimmune sera or sera from mice immunized with the unrelated Ab2, 4EA2-KLH was observed. Although steric hindrance by Ab3 binding
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FIGURE 2 Binding of polyclonal mice Ab3 sera to purified CEA by ELISA. The plate was coated with pure CEA (50 ng/well) and reacted with Ab3 sera (1/100 dilution) from mice immunized with Ab2 (3Ht) and unrelated Ab2 (4EA2) of the same isotype. F’reinmune sera and PBS-BSA were also used as controls. The results are presented as the mean OD at 405 nm 5 SEM; n = 6 for all groups. In a parallel control experiment, the same sera were checked on a plate coated with unrelated ovarian tumor glycoprotein. There was no reactivity, the OD value obtained was comparable to that obtained with PBS-BSA control. (Reproduced from the Journal of Immunology [29])
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Fluorescence FIGURE 3 Flow microfluorimetry analysis of LS174-Tcells with mice Ab3 sera. n m o r cells were reacted with Abl (A) and Ab3 sera (1/50 dilution) from mice immunized with Ab2, 3H1 (B). The reaction was developed with goat anti-mouse F(ab’)2 IgG-FITC labeled antibody. Re-immune mice sera were used as control (C). In D, human B cell lymphoma cells that do not express CEA were reacted with mice Ab3 sera raised against 3H1. Symbols used (-) Abl or Ab3 or preimmune sera, (. . .) PBS-BSA. (Reproduced from the Journal of Zmmunofogy [29]).
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cannot be excluded in these assays, the results suggest the presence of Ab3 antibodies that share idiotopes with Abl (8019). If the Ab2, 3H1 is a true internal image, then it should induce the production of Agspecific Ab3 in the absence of exposure to Ag in a genetically unrestricted way and across species barriers. To confirm this, we immunized rabbits with Ab2 3H1 for the production of Ab3 that might share idiotopes with Abl and exhibit identical binding specificity. The antisera from rabbits 729 and 730, immunized with 3H1, at 1/10dilution, inhibited binding of iodinated 8019 to Ab2 by 88 and 57% respectively. No significant inhibition was obtained with preimmune rabbit sera. It has been previously shown that mAb 8019 specifically immunoprecipitatesthe 180,OOO m.w. CEA by SDS-PAGE analysis [37]. To confirm that the Ab3 induced by 3H1 was specific for the CEA molecule, semipurified extract of CEA was separated by SDS-PAGE and transblotted to nitrocellulose filters. One filter strip (Fig. 4, lane 2) was stained with Buffalo black. There were two overlapping bands at the 180,000 m.w. region (CEA) and one major band at the 50,000 m.w. region (normal cross-reacting Ag) and a few minor low
FIGURE 4 Western blotting analysis of CEA extract with Abl and Ab3. Semipurified CEA extract was separated by SDS-PAGE and transblotted into nitrocellulose filters. Lane 1 (m.w. marker) and lane 2 (CEA ext) were stained with Buffalo black. The rest of the lanes were incubated with antibody as follows: lane 3 (8019 IgG1, 10 mg/ml), lane 4 (rabbit Ab3 sera against 3H1,1/500 dilution in PBS-BSA-Tween 20), lane 5 (preimmune rabbit sera, 11500 dilution in PBS-BSA-Tbeen 20) and lane 6 (control rabbit Ab3 sera against 4EA2, 1/500 dilution in PBS-BSATbeen 20). The reaction was developed by ELISA assay using goat anti-mouse immunoglobulin and goat antirabbit immunoglobulin reagents conjugated with alkaline phosphatase. (Reproduced from the Journal of Immunology ~ 9 1 ) .
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m.w. bands. The remaining filter strips were then incubated with mAb 8019, rabbit Ab3 sera, and control rabbit sera immunized with unrelated Ab2P 4EA2 (isotype matched). The reaction was developed by the ELISA assay. mAb 8019 (Fig. 4, lane 3) and rabbit Ab3 (lane 4) immunoprecipitated only molecules with a molecular mass of 180,000 Da from this complex mixture. The materials that were not precipitated by mAb 8019 or rabbit Ab3 sera, contained a wide range of lower m.w. CEA-related antigen. There was no reactivity with preimmune (Fig. 4, lane 5 ) or control sera (lane 6). The Western blotting analysis confirmed the specificity of mAb 8019 and the reactivity of rabbit Ab3 with 180,000 m.w. CEA. We have recently generated anti-anti-Id mAb from syngeneic mice immunized with 3H1 [29]. mAb Ab3 (culture supernatantdiluted 1/5) also immunoprecipitatedthe same 180,000 m.w. CEA as Abl by Western blotting analysis (Fig. 5). The idiotype-anti-idiotype interactions in the 8019 (Abl) system have been depicted in Figure 6. We compared the reactivities of Abl 8019 with that of Ab3 (polyclonal as well as monoclonal) by a sensitive immunoperoxidase assay on normal colon and colonic tumor specimens surgically removed from patients. The pattern of reactivity of Ab3 on both
FIGURE 5 Western blotting analysis of CEA with Abl and monoclonal Ab3 culture supernatant. Experimental details are same as in Figure 4. Lane 1 (8019 IgGl, 10 mglml), lane 2 (mAb culture supernatant, 1/5 dilution in PBS-BSA-Ween 20), lane 3 (control myeloma P3-653 cult supernatant). (Reproduced from the Journal of Immunology 1291).
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CEA
Ab' - A b 2 p (8019) (3H1)
Ab3 (=Abl')
FTGURE 6 Idiotype network for human gastrointestinal carcinoma
normal and malignant colonic tissues was almost identical to that obtained with Abl. There was no reaction with normal colonic mucosa. mAb 8019 and polyclonal mouse and rabbit Ab3 sera, as well as Ab3 mAb showed intense reactivity with colonic tumors. The staining was apical in gland-like structures and granular (cytoplasmic) in less differentiated areas. There were, however, minor, subtle differences between the staining patterns obtained with mAb Abl (IgG1, K ) and mAb Ab3 (IgM, K). Reaction with 8019 (Abl) resulted in the staining of tumor cells as well as secreted mucinous materials whereas reaction with mAb Ab3 resulted in the staining of tumor cells with no staining of secreted mucin [Fig. 71. Viale et al. [42] also observed minor differences in the staining patterns of Abl and Ab3 mAbs and hypothesized that these differences might reside in the nature of the immunogens used to raise the two mAbs (nominal antigen vs internal image). We plan to compare Abl and Ab3 mAb on a large panel of normal and malignant tissues. Collectively, these data suggest that such Ab2P may be useful as an antigen substitute for the induction of therapeutic immunity in CEA positive tumor patients.
DISCUSSION The anti-idiotype therapy approach has been tested and has shown to be effective in several animal tumor models [15, 43-45]. Particularly interesting are the observations that some but not all Ab2ps can induce protective immunity against tumor growth. Structure/function relationships would have predictive values in active immunotherapy using TAA-mimicking anti-Id. There exists very few anti-idiotype reagents to human cancer that have been characterizedserologically and biologically that show promise for clinical trials. To the best of our knowledge, 3H1 is the only potential anti-mAb reagent available for anti-idiotype immunization therapy of CEA positive GI cancer patients. The primary questions to be resolved are whether this anti-idiotypereagent can evoke an Ab3 as well as cellular immune response in patients; whether any Ab3 so derived behaves as an Abl-like antibody and whether this Ab3 and/or cellular immunity can mediate a potential antitumor effect. There
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FIGURE 7 Immunoperoxidase staining of colonic adenocarcinomas by monoclonal Abl and monoclonal Ab3. Serial sections were stained with a, 8019 IgGl (10 mg/ml, note intense staining in tumor area (T) and mucinous materials(M); b, Ab3 culture supernatant (neat), intense staining in the tumoral area (T)but no staining in the mucinous materials (M); c,P3-653 myeloma supernatant (neat), no reactivity in tumoral area (T) or mucinous materials (M). All sections were counterstained with hematoxylin X 240. (Reproduced from the Journal of immunology [29]).
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is evidence that certain Ab2 can induce an immune response in “silent clones” of lymphocytes which are not activated by mere exposure to nominal antigen [13, 141. Active immunization of patients with Ab2P might generate an Ab3 (Abl’) response which will hopefully be able to recruit human effector cells or complement to destroy the tumor by antibody-dependent-cell-mediatedcytotoxicity (ADCC) or complement-mediated cytotoxicity (CMC). In addition, anti-idiotype antibodies could induce various T cell activities such as stimulation of DTH and cytotoxic T cells specific for the tumor. Syngeneic monoclonal anti-idiotype antibodies have been successfully utilized as specific immunogens to elicit both T and B cell mediated immunity in our experimental L1210/GZL tumor system [19] and viral systems [lo, 461. In order to break tolerance to TAA, patients might have to be immunized with Ab2P after conjugation with a potential immunogenic carrier and mixed with an appropriate adjuvant. We expect to gather valuable information from these studies. Human therapeutic trials with anti-idiotypesare still in the very early stages. The knowledge gained from our attempts to modulate immune response of cancer patients with anti-idiotype reagents (3H1 for GI cancer and 4DC6 for T cell Ieukemiallymphoma)will help us to design similar studies with other human tumor systems. Since we have characterized a syngeneic monoclonal cascade of Abl, Ab2 and Ab3, starting with human CEA and gp37, these will enable us to analyze the structural basis of idiotypic antigen mimicry at the molecular level. This might help in the rational design of a more meaningful Hanti-idiotype vaccine in the future.
Acknowledgments This work was supported by United States Public Health Service Grants CA47860, CA51434. We thank Susan Morey and Zie Rue Zhang for excellent technical assistance and Pat Alvarado for typing of the manuscript.
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