Syngeneic Anti-Idiotype Monoclonal Antibodies to Murine Anticarcinoembryonic Antigen Monoclonal Antibodies'

(CANCER RESEARCH 49. 123-126, January 1, 1989] Syngeneic Anti-idiotype Monoclonal Antibodies to Murine Anticarcinoembryonic Antigen Monoclonal Antibodies'

Marc Monestier, Michael E. Debbas, and David M. Goldenberg2

Center for Molecular Medicine and Immunology, university of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103

ABSTRACT epithelia and in fetal colon (7). CEA is also detected in the sera of patients with other types of cancer, particularly if they are BALB/c mice were immunized with either NP-3 or MM, two anti- metastatic (7). CEA assays are used to monitor cancer patients, carcinoembryonic antigen murine monoclonal antibodies. Each animal produced anti-idiotype antibodies to the corresponding immunogen and and antibodies against CEA have been utilized successfully in no cross-reactivity between anti-NP-3 sera and anti-NP-4 sera was the radioimmunodetection of colorectal cancers (8). Various detected. Hybridomas were produced from these animals and two IgGi glycoproteins, immunologically related to CEA, have been re anti-idiotype monoclonal antibodies were obtained: CM1 specific for NP- ported to be present in normal tissues. The major members of 3 and CM11 specific for NP-4. CM1 and (Mil recognized determinants this family of CEA-related antigens include NCA, which shares located within the antibody-combining site, since each anti-idiotype an a similar tissue distribution with CEA and is also present on tibody inhibited the binding between the corresponding idiotype and granulocytes and macrophages, NCA-2 and NFA-2 found in carcinoembryonic antigens. Using an immunoblotting technique, neither meconium and adult feces, and the biliary glycoprotein I present CM1 nor CM11 reacted with isolated heavy or light chains of NP-3 or in normal bile (reviewed in Reference 7). CEA is a good NP-4, whereas binding was observed with the intact molecule. This observation indicates that CM1 and CM 11 are directed against confor- candidate for an idiotype vaccine since (a) it can be purified but mational idiotypes resulting from the association of the variable regions is not available in amounts sufficient for therapeutic purposes; of the heavy and light chains. Taken together, these results suggest that (b) CEA is a glycoprotein, therefore unlikely to be produced by CM1 and CM11 might bear internal images of carcinoembryonic antigen genetic engineering; and (c) the administration of monoclonal epitopes, and that they are potential candidates as idiotype vaccines anti-colorectal carcinoma antibodies had some positive effects against colorectal tumors. in patients with gastrointestinal tumors (1). Therefore, we initiated studies on the development of idiotype vaccines for CEA, and, in this report, we describe the production of anti- INTRODUCTION idiotype antibodies against NP-3 and NP-4, two anti-CEA Monoclonal antibodies directed against tumor-associated an monoclonal antibodies (9). tigens have been used in the therapy of human cancer (1). This approach is limited since the antibodies are administered pas MATERIALS AND METHODS sively and usually are of murine origin, leading to the produc tion of human anti-mouse immunoglobulins that can hamper Animals. 2 month-old BALB/c mice were purchased from HarÃan the efficacy of the treatment. An active immune response of the SpniÃÃiieDawleyLaboratories, Indianapolis, IN. patient toward the tumor would certainly be more efficient, but Monoclonal Anti-CEA Antibodies. NP-3 and NP-4 are two previously the immune system is usually tolerant to tumor-associated described anti-CEA monoclonal antibodies obtained from BALB/c antigens (2). The administration of a tumor-associated antigen mice (9). does not evoke a strong immune response since it is recognized Immunization Protocols and Monoclonal Antibody Production. as a self-component (3). Moreover, tumor-associated antigens BALB/c mice were immunized by i.p. injection of 100 ^g of purified NP-3 or NP-4 coupled to solubili/ed keyhole limpet hemocyanin by are rarely available in large purified amounts and their prepa glutaraldehyde fixation, and emulsified in complete Freund's adjuvant rations can be contaminated with viruses and viral products. As an alternative, anti-idiotype antibodies can be used as (Sigma, St. Louis, MO). Animals were boosted 7 days after the primary immunization and weekly thereafter (up to a maximum of five injec surrogate tumor-associated antigens (4). Idiotypes are antigenic tions) with 100 n% of the same immunogen in incomplete Freund's markers of the variable region of immunoglobulins, and it is adjuvant. The animals were sacrificed 4 days after the last injection and believed that interactions between idiotypes (AbI3) and anti- their splenocytes fused with the nonsecreting plasmocytoma SP2/0 idiotypes (Ab2) play an important role in the regulation of the according to a previously described technique (10). Culture superna- immune system (5). Certain anti-idiotype antibodies (termed tants were screened by ELISA for the presence of anti-idiotypes and Ab2/3)can mimic the three-dimensional structure of the original the positive hybridomas were cloned twice by limiting dilution. Isotypes antigen recognized by the Abl, and the administration of these were determined using the Screentype Kit (Boehringer Mannheim Ab2/3 (so-called idiotype vaccines) can trigger the production of Biochemicals, Indianapolis. IN). Monoclonal antibodies were purified from culture supernatants by affinity chromatography on Protein A- Ab3 antibodies recognizing this original antigen (6). Sepharose (Pharmacia, Piscataway, NJ). CEA is a complex glycoprotein (A/r 180,000) found mainly Sandwich Idiotype ELISA. This assay was used for the detection of in adenocarcinomas of entodermally-derived digestive system anti-idiotype antibodies in mouse sera and in culture supernatants as we described before (11). Briefly, the idiotype (NP-3 or NP-4) was Received 6/21/88; revised 9/21/88; accepted 9/30/88. The costs of publication of this article were defrayed in part by the payment diluted at 10 Â¿ig/mlincarbonate buffer, pH 8.6, and added (SO^I/well) of page charges. This article must therefore be hereby marked advertisement in to a polyvinyl chloride flat bottom-microtitration plate. After overnight accordance with 18 U.S.C. Section 1734 solely to indicate this fact. incubation at 4Â°C,wells were post-coated with 50 ^1 BSA (1% in PBS ' Supported in part by NIH Grant CA 39841. 2To whom requests for reprints should be addressed, at the Center for containing 0.05% Tween-20) for l h at room temperature. Culture Molecular Medicine and Immunology, 1 Bruce Street, Newark. NJ 07103. supernatants or mouse sera diluted in PBS-Tween-BSA were added (50 3The abbreviations used are: Abl. antibody directed against a conventional nl/well) and incubated for 2 h at room temperature. After washing, the antigen; Ab2, anti-idiotype antibody: Ab3. anti-anti-idiotype antibody: CEA, biotinylated idiotype (identical to the one used to coat the plate) was carcinoembryonic antigen; NCA. normal cross-reacting antigen: NCA-2, normal added at a concentration of 0.25 Â¿ig/mlin PBS-Tween-BSA. After cross-reacting antigen-2: NFA-2, normal fecal antigen antigen-2: ELISA, enzyme- linked immunosorbent assay; PBS. phosphate-buffered saline; BSA. bovine serum washing, avidin-peroxidase (Vector Laboratories, Burlingame, CA) was albumin. added at 0.25 Â¿ig/mlin PBS-Tween-BSA for l h at room temperature. 123

After extensive washings, 100 ^1 of substrate (o-phenylene-diamine 0.4 supernatants by affinity chromatography. To assess the speci mg/ml, H2O2, 0.012% in citrate-phosphate buffer, pH 5) was added to ficities of these anti-idiotype antibodies, both CM1 and CM 11 each well and the optical density was read at 450 nm using a kinetic were tested against a panel of monoclonal antibodies of various microplate reader (Molecular Devices, Palo Alto, CA). specificities belonging to the major immunoglobulin subclasses Direct Idiotype ELISA. This assay was used to assess the specificity of our purified anti-idiotype antibodies. Briefly, microtiter plates were (IgA, IgM, IgGi, IgG2a, IgG2b, IgG3. This was performed using coated with idiotype or other antibodies to be tested and post-coated a direct idiotype ELISA (results shown in Fig. 1, A and B). with BSA as described above. The biotinylated anti-idiotype was added CM1 appears to be exquisitely specific for NP-3 whereas CM11 at the concentration of 0.03 Mg/ml in PBS-Tween-BSA for l h at room shows a few cross-reactivities but binds preferentially to NP-4. temperature. After washing, avidin-peroxidase was added and the assay Inhibition of the Binding of NP-3 and NP-4 to CEA by CM1 conducted as described above. and CM11. Anti-idiotypes can bind to determinants located Inhibition of Binding to CEA by Anti-idiotype. This assay was con within the antigen-combining site of the antibody molecule or ducted to determine whether or not the anti-idiotypes bound to a to determinants located within the variable region but outside determinant located within the combining site of the idiotype-bearing of the combining site. A prerequisite for an Ab2/3 (internal antibody. Biotinylated NP-3 or NP-4 (1 Mg/ml in PBS-Tween-BSA) image type) is its ability to bind within the combining site and was incubated overnight with various concentrations of anti-idiotype or of an irrelevant isotype-matched monoclonal antibody. The mixture therefore to inhibit the binding between the Abl and the anti was then transferred onto CEA-coated microtiter plates for half an gen. The data in Fig. 2 show that CM1 inhibited the binding of NP-3 to CEA but not the binding of NP-4. Conversely, CM11 hour at room temperature and, after washing, the assay was pursued with avidin-peroxidase as described above. inhibited the binding of NP-4 to CEA but not the binding of Immunoblotting of NP-3 and NP-4 Monoclonal Antibodies. This assay NP-3. As a control, an irrelevant isotype-matched monoclonal was carried out to determine whether or not the idiotypes recognized IgGiK (MOPC21) did not affect the bindings of NP-3 and NP- by our monoclonal antibodies were detected on isolated heavy or light 4. chains of NP-3 and NP-4. It was conducted as we described before with Immunoblotting of NP-3 and NP-4 using Monoclonal Antiidi- slight modifications (11). Briefly, NP-3 and NP-4 were reduced with otypes. Idiotypes can be present on the isolated heavy or light 5% 2-mercaptoethanol and their heavy and light chains separated in a chains of immunoglobulins or result from the association of 10% polyacrylamide-sodium dodecyl sulfate gel and transferred onto nitrocellulose using a Mini Trans-Blot Cell (Biorad, Richmond, CA). both chains (reviewed in Reference 12). After reduction, the As controls, unreduced NP-3 and NP-4 were resolved in a 5% poly heavy and light chains of NP-3 and NP-4 were electrophoreti- acrylamide-sodium dodecyl sulfate gel and blotted as above. Control cally separated and transferred onto nitrocellulose. None of the nitrocellulose strips were stained with Amido Black. Blots to be reacted two monoclonal anti-idiotype antibodies reacted with isolated with monoclonal anti-idiotypes were blocked for l h with BSA (1% in Tris-buffered saline containing 0.05% Tween-20). The reactions with 0.7 the biotinylated anti-idiotypes were conducted similarly to the direct idiotype ELISA described above except that Tris-buffered salini- 0.6 CM 1 Tween-BSA was used as a diluent and the substrate was diaminoben- zidine (5 mg/ml) in Tris-buffered saline containing 0.015% H2Ã›2. E 0.5--

f 0.4- RESULTS Specificity of the Anti-idiotype Response. BALB/c mice were immunized repeatedly with either NP-3 or NP-4, and their sera o 0.2-â€¢ were assayed for anti-idiotype activity. The data in Table 1 O show that no Ab2 were detected prior to immunization and 0.1 -- that, after immunization, the anti-idiotype response was ex 0.0 tremely specific, since the mice immunized with NP-3 produced NP3 NP4 MU9 -MOPC- Y5606 BA6 Py216 anti-NP-3 Ab2 but no anti-NP-4 Ab2. Conversely, the animals 104E 21 315 injected with NP-4 produced Ab2 specific for NP-4 even though both NP-3 and NP-4 are IgG,K. Generation of Anti-idiotype Monoclonal Antibodies. Hybrid 0.30 orna supernatants from mice immunized with NP-3 or NP-4 B 0.25-- were screened using a sandwich idiotype ELISA. After cloning ESS CM 11 of the positive hybridomas, two monoclonal anti-idiotypes, CM1 binding to NP-3 and CM 11 binding to NP-4, were ob 0.20- O tained. Both antibodies are Igd/c and were purified from culture m 0.15-- Table 1 Specificity ofsyngeneic anti-idiotype sera :' The anti-idiotype activity was measured by a sandwich idiotype ELISA, as O 0.10-- described in the "Materials and Methods" section. O

BALB/c immunized immunized 0.05-- NP-3"Preimmunization with withNP-40.025

PostimmunizationrPreimmunization 0.00 0.2460.044 0.0240.019 assayAnti-NP-4 NP3 NP4 MU9 -MOPC- Y5606BA6 Py216 104E 21 315 Postimmunization0.045* 0.040BALB/c 0.323Anti-NP-3 assay Fig. 1. Anti-idiotype specificities of CM1 (A) and CM 11 (B). Binding of "The assay was performed with a 1/100 dilution of the sera of animals biotinylated CM1 and CM 11 (0.03 //g/'nil) to various monoclonal proteins was immunized with NP-3 or NP-4. determined using a direct idiotype ELISA. The isotypes of these monoclonal * Values represent OD45onmafter background subtraction. proteins are NP-3: IgG,Â«, NP-4: IgG.n, MU9: IgdÂ«, MOPC104E: IgMX, c After three injections of idiotype. MOPC21: IgG.K, Y5606: IgG3X, MOPC315: IgAX, BA6: IgG2bX,Py216: IgG2,/c. 124

120 DISCUSSION 100- .A The ultimate goal of our project is to trigger, in patients with 80- gastrointestinal carcinomas, an antitumor immune response using anti-idiotype antibodies carrying the internal image of 60 tumor antigens. We describe, in this communication, the first 40- step of this endeavor: the generation of two syngeneic anti- 20-- idiotype monoclonal antibodies, CM1 and CM11, against mu rine monoclonal anti-CEA antibodies. Because of the presence TJ C of CEA-related antigens in normal tissues, it is necessary to OD generate an Ab2/3 carrying the internal image of a CEA-specific 100- Ã ? B epitope. Indeed, immunization against a cross-reactive epitope 80- could trigger a potentially harmful autoimmune reaction. Therefore, we chose NP-3 and NP-4, two anti-CEA monoclonal 60- Inhibitor OMOPC21 antibodies that do not cross-react with antigens present in 40- ACM 1 normal adult tissues. In previously published experiments (9), â€¢CM11 it was shown that neither NP-3 nor NP-4 reacted with NCA. 20- Assay run with NP4 (â€¢ ) or NP3 ( ) On the other hand, NP-3 (but not NP-4) reacted with meconium-derived antigen, a meconium-purified antigen similar, if 0.02 0.05 0.10 0.20 0.50 1.00 2.00 5.00 10.00 not identical, to NCA-2 (14). None of these antibodies bound Inhibitor Concentration (Â¿Â¿g/ml) granulocytes,4 which can be recognized by anti-CEA antibodies Fig. 2. Inhibition of the binding of anti-CEA monoclonal antibodies to CEA (15). Moreover, both NP-3 and NP-4 have been utilized for the by anti-idiotype monoclonal antibodies. I, CM1 inhibits the binding to CEA of radioimmunodetection of CEA-producing tumors in human NP-3 but not of NP-4. B, CM 11 inhibits the binding to CEA of NP-4 but not of cancer patients, without showing any cross-reactivity with nor NP-3. No inhibition was observed with a control antibody (MOPC21). mal tissues.4 Only a fraction of the anti-idiotype population carries the internal image of an external antigen (Ab20), but there is no means to direct the anti-idiotype response preferentially to wards the internal image-bearing Ab2/3 (6). Prior to in vivo administration of Ab2, only immunochemical characteristics can provide clues about the presence of an internal image. After having assessed the anti-idiotype nature of CM1 and CM11, we demonstrated that each Ab2 was able to inhibit specifically the binding of the corresponding idiotype to CEA. This obser vation is crucial with respect to the Ab2 nature of CM 1 and CM11, since both CEA and an Ab2 carrying the internal image of CEA must bind to the same determinants within the Abl- combining site. Moreover, our observation by the immunoblot ting technique that both heavy and light chains of the idiotype are needed for the Abl-Ab2 interaction is another argument

REDUCED NP3 NP4 supporting the presence of internal images of CEA epitopes on CM1 and CM11. Administration of these two Ab2s to synge A B UNREDUCED neic animals, and the detection of Ab3 with specificity for the original antigen (CEA), will enable us to answer this question. Fig. 3. Immunoblotting of NP-3 and NP-4 using anti-idiotype monoclonal The anti-idiotype approach to synthetic vaccines has stimu antibodies. Neither CM1 nor CM 11 bind to isolated heavy and light chains of NP-3 or NP-4 (A). CM1 binds to intact, non-reduced NP-3 and CM11 binds to lated numerous studies using Ab2 carrying the internal image intact, non-reduced NP-4 (B). of microbial or tumor antigens (reviewed in References 6 and 16). In several animal models, Ab2s have been used successfully heavy or light chains of NP-3 or NP-4 (Fig. 3A). As a control, to trigger the immune system, exerting protective effects against when NP-3 or NP-4 were not reduced but electrophoretically various microbial antigens: Trypanosoma rhodesiense (17), resolved and blotted as above, CM1 reacted with NP-3 and Schistosomia monsoni (18), Hepatitis B surface antigen (19), CM11 reacted with NP-4 (Fig. 35). These findings strongly Streptococcus pneumoniae (20), etc. Similarly, Ab2s carrying the internal image of tumor antigens induced protective im suggest that both CM1 and CM 11 recognize an idiotype re munity against the corresponding tumor: mice injected with sulting from the association of the heavy and light chains. It is 2F10, an Ab2 carrying the internal image of an epitope common nevertheless possible that an idiotype present on a single chain is destroyed during the reduction with 2-mercaptoethanol, but to mouse mammary tumor virus and to the L1210/GZL tumor, we and others have observed that single chain-idiotypes are were protected against a subsequent challenge with L1210/ GZL (21). The administration of HIM/1/230, an Ab2 carrying usually detected by immunoblotting (11, 13). The recognition the internal image of a rat sarcoma, also exerted protective of such a conformational idiotype is important since, in most effects against the growth of the tumor (22). It was also dem cases, the binding of the Abl to the antigen depends on the onstrated in several systems that animal Ab2/3 can carry the association of both heavy and light chains. Therefore, an Ab2 internal image of human tumor antigens (23-25). An "idiotype binding to an idiotope necessitating both heavy and light chains vaccine" trial has been reported in human cancer patients (26), is a more likely candidate to bear the internal image of the original antigen. ' D. M. Goldenberg, unpublished data. 125

tration of anti-idiotypic antibodies in mice. Proc. Nati. Acad. Sci. USA, 81: where patients with advanced colorectal carcinomas were 2864-2867, 1984. treated with alum-precipitated polyclonal goat anti-idiotypic 5. Jerne, N. K. Towards a network theory of the immune system. Ann. Immunol. antibodies to a murine monoclonal anti-colorectal carcinoma (Paris), 125C: 373-389, 1974. . 6. Bona, C., and Moran, T. Idiotype vaccines. Ann. Immunol. (Paris), 136C: antibody. All patients developed Ab3 binding to the surfaces of 299-312, 1985. cultured tumor cells, but the therapeutic efficacy of such a 7. Rogers, G. T. Carcinoembryonic antigens and related glycoproteins. Molec protocol remains to be established. Recently, Ziai et al. (27) ular aspects and specificity. Biochim. Biophys. Acta, 695: 227-249, 1983. 8. Goldenberg, D. M. Tumor imaging with monoclonal antibodies. J. 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In most experimental systems, Ab2 has been heterogeneity of Carcinoembryonic antigen: purification from meconium of an antigen related to Carcinoembryonic antigen. Cancer Res., 43: 679-685, administered before the challenge with the tumor and this does 1983. not parallel the clinical conditions, where the diagnosis of the 15. Dillman, R. O., Beauregard, J. C., Sobol, R. E., Royston, I., Bartholomew, R. M., Hagan, P. S., and Halpern, S. E. Lack of radioimmunodetection and tumor would precede the therapeutic Ab2 injection. It remains complications associated with monoclonal anti-carcinoembryonic antigen to be determined whether Ab2 given to animals or patients with antibody cross-reactivity with an antigen on circulating cells. Cancer Res., detectable tumors can be effective. The term "idiotype stimu â€¢Â«.â€¢2213-2218,1986. lation" would be more appropriate than "idiotype vaccine" in 16. Dreesman, G. R., and Kennedy, R. C. 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Marc Monestier, Michael E. Debbas and David M. Goldenberg

Cancer Res 1989;49:123-126.

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