Induction of Antigen-Specific Immune Response with Use of Anti-Idiotypic Monoclonal Antibodies to Anti-Carcinoembryonic Antigen Antibodies1

(CANCER RESEARCH 51, 2599-2604, May 15, 1991] Induction of Antigen-specific Immune Response with Use of Anti-Idiotypic Monoclonal Antibodies to Anti-Carcinoembryonic Antigen Antibodies1

Masayuki Tsujisaki, Kohzoh linai, Shigeru Tokuchi, Yoshiaki Hanzawa, Tadao Ishida, Hiroshi Kitagawa, Yuji Hinoda, and Akira Yachi2

Department of Internal Medicine (Section 1), Sapporo Medical College, S-l, W-16, Chuo-ku. Sapporo, 060 [M. T., K. I., S. T., T. I., Y. H., A. Y.], Life Sciences Laboratories, Ajinomolo Co., Yokohama {Y. H.], Department of Basic Research, Hoechst Japan Limited, Kawagoe, Saitama [H. K.], Japan

ABSTRACT Id MoAb (Ab2) to MoAb (Abl) reacting with tumor-specific transplantation antigen of rat sarcomas. When the Ab2 were Anti-idiotypic monoclonal antibodies (MoAbs) were prepared in a given to rats, they protected against challenge with the tumor. syngeneic system against anti-carcinoembryonic antigen (CEA) MoAb Kennedy et al. (8) reported that a combination of sera, which 5B3 (IgGl), which reacted with a carbohydrate moiety on CEA, and contained Ab2 relating to the amino-terminal and the carboxy- MoAb MA208 (IgGl), which reacted with a peptide on CEA. Anti- idiotypic MoAb T3-503 and T4-202 recognized the private idiotype of terminal peptides of the SV40 T-antigen, induced resistance in MoAb 5B3; anti-idiotypic MoAb M7-049 and M7-625 did so for MoAb mice to challenge with syngeneic SV40-induced tumor cells. As MA208. Idiotype mapping showed that MoAb 5B3 has at least two expected from studies on animal tumors, Ab2 could have been distinct idiotopes at its combining site and MoAb MA208 also has two. generated, relating to human cancer cells. Nepon et al. (9), for Four different anti-idiotypic MoAbs (Ab2) could induce anti-anti-idi- the first time, prepared polyclonal Ab2 by immunizing rabbits otypic antibodies (Ab3) specific to their respective immunizing anti- against a mouse anti-A/r 97,000 MAA MoAb 8.2. Recently, idiotypic MoAbs. Anti-anti-idiotypic MoAb M7-625 antiserum (at least Kahn et al. (10) demonstrated that some of monoclonal Ab2 a part of the antibodies) have the same reactivity as MoAb MA208, induced an Abl-like Ab3 response when given to either synge since the serum competed with the binding of MoAb MA208 against neic or allogeneic mice. CEA and contained the antibody population reactive with purified CEA in immunoblotting assay. These results suggest that anti-idiotypic MoAb We have carried out idiotype analysis by using anti-Id anti M7-625 bears the internal image of the antigen (CEA) and induces the bodies to anti-human high-molecular-weight MAA MoAbs (11, anti-anti-idiotypic antibodies specific to CEA. Therefore, an anti-idiotypic 12) in a syngeneic system. The results suggest that: (a) anti- antibody bearing the internal image of a tumor associated antigen might MAA MoAbs (Abl) were able to induce polyclonal anti-Id be used as a possible tool for vaccination or immunotherapy against antibodies (Ab2) specific to corresponding anti-MAA MoAb in malignant tumors as an antigen specific immunomodulator. a syngeneic mouse system; (b) polyclonal anti-Id antibodies inhibited the binding of anti-MAA MoAb to MAA on cultured melanoma cells, suggesting that the polyclonal anti-Id antibod INTRODUCTION ies included a population of anti-Id antibodies reactive with a The idiotype network theory proposed by Jerne (1) has been combining site of anti-MAA MoAb; (c) idiotype mapping uti accepted as a basic concept of immune regulation. This hypoth lizing anti-Id MoAbs showed that there might be several distinct esis of immune regulation proposes that idiotype-anti-idiotype idiotopes at combining and noncombining sites; (d) these anti- reactions control the response of a host to an antigen according Id MoAbs (Ab2) could also induce their anti-anti-Id antibodies to either an enhancing or suppressing feedback mechanism. (Ab3) specific to the corresponding immunizing anti-Id MoAb Numerous studies (2, 3) have acknowledged that idiotype net (Ab2) when the Ab2 was immunized in syngeneic BALB/c works have important roles in regulation of the immune mice. response. In the system of these human melanoma-associated antigens, In addition, Id3 antibodies have made it possible to analyze it has been very difficult to conclude clearly that anti-Id MoAb the genesis of autoimmune diseases and to perform structural carries the internal image of a given antigen, since the molecular analysis of receptors and ligands in receptor diseases (4). Re structure of MAA is not analyzed definitely. Recently, molec cently, Mendlovic et al. (5) reported that C3H mice developed ular cloning of CEA, one of the most useful tumor markers for the immunocomplex nephritis after injection with a human human colorectal carcinoma, was carried out and structural monoclonal anti-DNA antibody bearing Id-16/6. The animals analysis was developed. In order to investigate the internal developed not only high levels of anti-Id-16/6 but also anti- image from the molecular aspects and to determine what kind (anti-Id-16/6) antibodies that had anti-DNA activity. Also, anti- of anti-Id antibody is useful for vaccination or cancer therapy, Id antibody has successfully been applied as a vaccination to B- we have attempted to prepare a set of anti-Id MoAb relating to type viral hepatitis in animals (6). CEA. In experimental tumor system, Dunn et al. (1) generated anti- MATERIALS AND METHODS Received5/21/90;accepted3/7/91. The costs of publication of this article were defrayed in part by the payment Preparation of Purified CEA and NCA. Purified CEA and NCA of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (nonspecific cross-reacting antigen) were prepared from colonie cancer 1Supported by grants for Cancer Research from the Ministry of Education, tissues and from normal lung tissues, respectively, by the immunoper- Science and Culture to A. Yachi and K. Imai, and from the Ministry of Health chloric extraction method followed by successive gel filtration as de and Welfare, Japan, to A. Y. This work was also supported and encouraged by scribed elsewhere (13). Purified CEA and NCA were used as antigens the Incitement Award of the Japanese Cancer Association to K. I. 2To whom requests for reprints should be addressed, at Department of Internal in the binding assay and in the immunoblotting assay. Medicine (Section 1), Sapporo Medical College, S-l, W-16. Chuo-ku, Sapporo Animals. Eight- to 12-week-old female BALB/c mice were purchased 060 Japan. 3The abbreviations used are: MoAbs, monoclonal antibodies; CEA, carcinoem- from Charles River Breeding Laboratories, Wilmington, MA. bryonic antigen; NCA, nonspecific cross-reacting antigen; Id, idiotypic; PBS, Development of Monoclonal Antibodies to CEA. The monoclonal phosphate-buffered saline; MAA, melanoma-associated antigen; PBS-T20, PBS antibodies were secreted by hybridomas constructed with myeloma cells containing 0.05% Tween 20. and splenocytes from BALB/c mice which were immunized with 3 2599

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1991 American Association for Cancer Research. ANTl-IDIOTVPIC MoAbs TO ANTI-CEA Abs weekly i.p. injections of 1 x IO7CEA-producing pancreatic carcinoma bound radioactivity was counted in a gamma counter. Results were cells Panel or 100 ^g of purified CEA. Hybridization with murine expressed as the percentage of binding inhibition as compared with myeloma cells P3x63-Ag 8.653, subcloning and growing of hybridomas control. were done according to standard procedures (13). Monoclonal antibod Idiotype Binding Assay. 125I-Labeled anti-idiotypic MoAb (1 x 10' ies 5B3 (IgGl) and MA208 (IgGl) had strong reactivity to purified cpm) were incubated for 3 h at 4Â°CwithMoAb-coated microtiter plates, CEA and CEA-producing cultured cells (MKN45, KATOIII, Panel, using the same procedure as in the sandwich assay. The plates were and WiDr), but neither reacted to purified NCA, meconium antigens, washed 5 times with PBS-T20, wells were cut, and the radioactivity nor CEA-nonproducing cells (K562 and Daudi) in binding assay. MoAb was counted in a gamma counter. 5B3 recognizes an epitope of carbohydrate moiety on CEA and MoAb Association constants (A,) of monoclonal antibodies were measured MA208 recognizes that of a peptide on CEA (14). according to Scatchard analysis (25). Briefly, 100 n\ of serial diluted Other Monoclonal Antibodies. The anti-CEA MoAbs, other than 5B3 '"Â¡-labeled anti-Id MoAbs (1.56 x 10~8~ 4.87 x IO'10 M) were added and MA208, were generous gifts from research groups of the Interna to the plates coated with MoAb MA208. Following 5 washings, bound tional Workshop (15). This Workshop on the epitope reactivity of cpm were counted. B/F and [B] were plotted to calculate A"aaccording MoAbs against CEA has summarized that anti-CEA MoAbs have been to the slope of the line [B, cpm of bound antibodies; F, cpm of free divided into 5 groups according to serological studies, mainly compe antibody; [B], concentration of bound antibody (M)]. tition assay. The studies indicate that anti-CEA MoAbs CEA 281 and Cross-blocking Assay. Microtiter plates were coated with MoAb 26/5/1; CEA 41 and 12-140-7; T84-66, II 10, and 12-140-1 were in (Abl) by incubating for 16 h at 4Â°C.After washing, an excess of cold the same groups, respectively ( 15). On the other hand, anti-CEA MoAbs anti-idiotypic MoAbs (Ab2) were added and incubated for 3 h at 4Â°C. YK024 and MT008 recognize the carbohydrate moiety on CEA (13). Following 3 washings, '"I-labeled anti-idiotypic MoAbs (1 x IO5cpm/ MoAb MA503 and MA504 recognize NCA which is a member of the well) were added to test for their ability to bind. After incubation for 4 CEA gene family. MoAb VH206 recognizes asialo glycoprotein which h, plates were washed 5 times and bound radioactivity was counted. belongs to a high-molecular-weight mucin, with M, > 500,000 (16), Results were calculated as percentage of blocking, compared with while MoAb MUSEI 1 is against the peptide epitope in a M, 300,000 negative control. mucin molecule (17), and MoAb BM2 recognizes X-hapten.4 The Induction of Syngeneic Anti-Anti-Idiotypic Antibodies by Anti-idi MoAbs to human MAA include the anti-high-molecular-weight mela otypic Monoclonal Antibodies. BALB/c mice were immunized by an i.p. noma-associated antigens MoAb 149.53, 225.28, and 763.74 that rec injection of 100 /ig of purified anti-idiotypic MoAb coupled to solubi- ognize distinct epitopes on the same molecule (11. 18), and anti-A/r lized keyhole limpet hemocyanin by glutaraldehyde fixation and mixed 96,000 MAA MoAb CL203 that recognizes intercellular adhesion with complete Freund's adjuvant (11). Animals were bled 3 days after molecule 1 (ICAM1) (19). The anti-HLA class I MoAbs include anti- the last booster and their antisera were used in the analysis. HLA-A2, A28 MoAb CR11-351 and KS1, and the MoAb CR11-115 Sandwich Assay and Inhibition Assay to Detect Anti-Anti-Idiotypic to monomorphic determinants (12). The anti-HLA class II MoAbs Antibodies. Sandwich assay was performed by a procedure similar to include the anti-HLA-DR, DQ, DP MoAb Q 5/13, and anti-HLA-DR, that described above. Briefly, microtiter plates were coated with anti- DP MoAb Q2/80 to monomorphic determinants (20). idiotypic MoAbs, and then 100 M'of diluted sera (1:10) were added and Development of Anti-Idiotypic Monoclonal Antibodies. Anti-idiotypic incubated for 4 h. Following 3 washings, plates were incubated with monoclonal antibodies were prepared against murine anti-CEA MoAbs '"I-labeled anti-idiotypic MoAb for 4 h. Plates were washed 5 times in a syngeneic system, using the methods as described before (11, 12). and the radioactivity was counted. Briefly, hybridomas were constructed with splenocytes from a BALB/ Inhibition assay was done by the modified procedure. Fifty n\ of 125I- c mouse primed with 100 ng of purified murine anti-CEA MoAb labeled anti-idiotypic MoAbs (1 x IO5cpm) were incubated with 50 p\ coupled to keyhole limpet hemocyanin and polymerized with glutaraldehyde in complete Freund's adjuvant (GIBCO Laboratories, Detroit, of diluted antisera (1:10) for 2 h. The mixture was added to an anti- idiotypic MoAb-coated plate and incubated for 2 h. Following 5 wash MI) (12). Seven and 14 days later, the mouse was boosted with the same immunogen in incomplete Freund's adjuvant. Three days after ings, bound radioactivity was counted and results were calculated as percentage of inhibition of binding as compared with control. the last booster, the mouse was sacrificed, the spleen was removed, and Competition Assay. Microtiter plates were coated with purified CEA the splenocytes were hybridized with murine myeloma cells P3x63-Ag (5 Mg/ml) by incubating for 16 h at 4Â°C.After washing, 50 n\ of serial 8.653 according to the procedure described above. diluted anti-anti-idiotypic sera (Ab3) generated with anti-idiotypic Purification and Radiolabeling of Monoclonal Antibodies. Monoclonal MoAb (Ab2) and 50 Mlof '"I-labeled MoAb (Abl, 1 x IO5cpm) were antibodies from the resulting hybridomas were purified from ascitic added and incubated for 2 h at 4Â°C.Plates were washed 5 times and fluid either by caprylic acid precipitation (21), by affinity chromatography on Protein A-Sepharose (22), or by ion exchange chromatography the radioactivity was counted. Results were calculated as percentage of on DEAE-cellulose. Antibodies were radiolabeled with '"I, utilizing competition compared with that of normal sera. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and either the chloramine-T method (23) or the lodogen method. Western Blotting. Purified CEA was separated by sodium dodecyl Sandwich Assay. The sandwich assay, as described before (24), was sulfate-polyacrylamide gel electrophoresis on 7.5% polyacrylamide slab performed as follows; 96-well polyvinyl chloride microtiter plates (Dy- gel, as described elsewhere (26). Transfer to nitrocellulose membranes natech, Alexandria, VA) were coated with a MoAb by incubating 100 was achieved, essentially as described by Towbin et al. (27), in a julof a MoAb solution ( 100 Mg/ml) in 0.1 Mbicarbonate buffer, pH 9.5, for 16 h at 4Â°C.Following 3 washings with PBS, pH 7.4, containing transblotting chamber (Bio-Rad Laboratories, Richmond, CA) at 170 PBS-T20,100 M!of hybridoma supernatants were added and incubation mA overnight, using 25 mmol/Iiter Tris and 192 mmol/liter glycine in was continued for 4 h at 4Â°C.The plates were washed 3 times with 20% methanol, pH 8.3. Nitrocellulose membranes were then sliced PBS-T20, the wells were cut, and the radioactivity was counted in a vertically into strips. These were incubated in 3% bovine serum albumin gamma counter. Nonspecific binding was measured by replacing anti- (Sigma) in PBS at room temperature for 6 h. Membranes were incu bated with diluted anti-anti-Id sera (1:50), preimmune serum (1:50) or idiotypic antibodies with irrelevant antibody. Specifically bound radio anti-CEA MoAbs (5 Mg/ml) for 2 h and then washed 3 times with PBS. activity was measured by subtracting nonspecifically bound cpm from Each strip was then incubated with the sera (dilution of peroxidase- total bound cpm. Inhibition Assay. Fifty pi of '"I-labeled monoclonal antibodies (1 x conjugated rabbit anti-mouse immunoglobulins) (Dako, Denmark). IO5 cpm) were incubated for 2 h at 4Â°Cwith 50 M' of hybridoma After being washed as above, the strips were incubated in a freshly supernatants. Then the mixture was added to cultured MKN45 CEA- prepared mixture of 60 mg of chloronaphthol in 20 ml of methanol producing gastric carcinoma cells (2 x I05/well). At the end of the 2-h and 60 Ml of H2O2/100 ml of 20 mivi Tris, pH 7.5. After a 15-min incubation at 4Â°C,target cells were washed 5 times with PBS, and the incubation at room temperature, the strips were removed into water. Antigen-antibody binding was identified by the presence of a black 4 Unpublished observation. stain. 2600

Table Characteristics ofanti-idiotypic monoclonal antibodies to anti-CEA RESULTS monoclonal antibodies 5B3 and MA208 assay" assay692 Development and Characterization of Anti-Idiotypic Monoclo MoAbT3-503T4-202YH206MUSEI(cpm)9,732 nal Antibodies to MoAb 5B3 and MoAb MA208. Syngeneic A.B.Anti-Id Â±872f9.271 Â±389 anti-Id monoclonal antibodies were prepared to anti-CEA Â±921320 Â±20Â± Â±52331 11 MoAb 5B3 and MA208. The cell fusion experiments resulted 1M7-049M7-625YH206MUSEllSandwichÂ±4110,242 Â±198 in 19 of 887 (2.1 %) clones to MoAb 5B3 and 13 of 962 (1.4%) clones to MoAb MA208, whose supernatants showed strong Â±72411.128 Â±296 +842354 Â±30Â± reactivity in sandwich assay and inhibition assay. From them, Â±33346 10Â± four MoAbs, designated T3-503, T4-202, M7-049, and M7- Â±28Inhibition 1 625 were established and used in this study. The sandwich assay " Microtiter plates coated with MoAb 5B3 (A) or MoAb MA208 (B) were incubated with hybridoma supernatants. After being washed. '!'I-labeled MoAb represents the binding activity of supernatants (candidates for anti-idiotypic MoAb) to MoAbs 5B3 and MA208, and the 5B3 (A) or MA208 (B) was added to test for binding (values denote mean Â±SD). *The mixture of '"1-labeled MoAb 5B3 (A) or MA208 (B) and hybridoma inhibition assay demonstrates the inhibitory activity of the supernatants was added to cultured CEA-producing cells. After being washed, the supernatant against the binding of '"I-labeled MoAb 5B3 or bound radioactivity was counted. The results are expressed as percentage of inhibition, compared with that of irrelevant MoAbs (values denote mean Â±SD). MoAb MA208 to CEA. Representative results are shown in ' Anti-mucin MoAb VH206 and MUSEI I are used as controls. Table 1. Anti-idiotypic MoAbs T3-503 and T4-202 recognized MoAb 5B3, while M7-049 and M7-625 recognized MA208. Control MoAbs YH206 and MUSE11 did not bind either Table 2 Specificity ofanti-idiotypic monoclonal antibodies elicited with anti- CEA MoAb 5B3 and MA208" MoAb 5B3 or MA208. MoAbs T3-503 and T4-202 remarkably inhibited the binding of MoAb 5B3 to CEA, and MoAbs M7- MoAbMoAb5B3MA208010CEA281CEA '"I-labeled anti-idiotypic 049 as well as M7-625 inhibited the binding of MoAb MA208 7-6251.259.90.60.70.70.60.50.70.60.60.70.60.60.60.70.60.40.50.60.50.40.50.60.50.60.5to CEA, whereas control MoAbs failed to inhibit. The specificity of these anti-Id MoAbs was determined by idiotype binding assay with the use of a variety of MoAbs. As shown in Table 2, each anti-Id MoAb to MoAb 5B3 or MA208 41T recognized the private idiotype, since it did not react with a 84-66II 1026/5/112-140-112-140-7YH024MT008MA503MA504YH206MUSEI panel of MoAbs to distinct epitopes of CEA, to NCA, to mucin, to high-molecular weight MAA, to HLA-class I antigen or to HLA-class II antigen, but reacted only with whichever corre sponding MoAb 5B3 or MA208 was used as the immunogen. The results presented in Tables 1 and 2 suggest that anti-Id MoAbs T3-503 and T4-202 specifically recognize idiotopes at the combining site of MoAb 5B3, and that anti-Id MoAbs M7- 1BM2149.53225.28763.74CL203CRll-115CRI 049 and M7-625 do so for MoAb MA208. Idiotype Mapping. In order to analyze the spatial relationship among idiotopes on MoAb 5B3 and MA208, a cross-blocking assay was done with use of anti-Id MoAbs. As shown in Table 96.000MAAHLA-class 1HLA-A2.A28HLA-A2.A28HLA-DR.DQ.DPHLA 3, anti-Id MoAb T3-503 and T4-202 to MoAb 5B3 failed to 1-351KSIQ5/13Q2/80SpecificityCEACEACEACEACEACEACEACEACEACEACEACEANCANCAMucinMucinX-haptenHMW-MAAHMW-MAAHMW-MAAM,affect the reactivity of the other antibody. Anti-Id MoAb M7- 049 and M7-625 inhibited the binding of the other MoAb to DR.DPT3-50333.5*0.90.80.90.60.50.70.80.90.50.80.60.70.80.70.60.60.60.60.50.50.60.70.60.70.7T4-20218.41.00.9.5.5.5.4.9.5.7.20.80.80.71.00.81.21.21.41.21.61.21.01.21.00.9M7-0491.180.70.40.40.40.40.30.40.30.40.40.40.40.50.40.40.30.40.40.40.30.40.40.40.40.3MMoAb MA208. The affinities of MoAb M7-049 and M7-625 Â°I25l-Labeled monoclonal anti-idiotypic antibodies were incubated for 3 h at were similar since the association constants (A"a)were 4.5 Â±0.5 4Â°Cwith MoAb-coated microtiter plates. Then the plates were washed and bound x IO7 (SD) and 3.2 Â±0.3 x IO7 M~', respectively. Therefore, radioactivity was counted in a gamma counter. * Results are expressed as cpm x IO3. these results suggest that there were at least two distinct idi otopes (spatially far) at the combining site of MoAb 5B3 and two distinct idiotopes (overlapping) at that of MoAb MA208, respectively. Schematic figure is shown in Fig. 1. Induction of Anti-Anti-Idiotypic Antibodies. Immunization with four distinct anti-Id MoAbs T3-503 and T4-202 to MoAb Table 3 Idiotopes recognized with anti-idiotypic monoclonal antibodies to anti- CEA MoAb SB3 and MA208" anti- InhibitorA. MoAbT3-50391

Combining MoAbT3-503 Â±5* 2 site MoAbT4-202B. 6Â±2M7-04996 87Â±4M7-62590

MoAbM7-049 Â±551 Â±5 MoAb M7-625'2!I-Labcled Â±3idiotypicT4-2020Â±96 Â±4 â€¢Microtiter plates coated with MoAb 5B3 (A) or MoAb MA208 (B) were incubated with an excess of cold anti-idiotypic monoclonal antibodies and then tested for their ability to bind '"I-labeled anti-idiotypic monoclonal antibodies. MoAb 5B3 MoAb MA208 * The results are expressed as percentage of inhibition (values denote mean Â± Fig. 1. Mapping of idiotopes at the combining site of anti-CEA monoclonal SD). antibodies 5B3 and MA208. 2601

Table 4 Analysis of anti-anti-idiotypic anlisera generated with monoclonal anti- that anti-anti-Id MoAb M7-625 antiserum had a reactivity to idiotypic antibodies to anti-CEA MoAb 5B3 and MA208 purified CEA, while other antisera did not (Fig. 3). Anti-anti- antiserumanti-idiotypicMonoclonalAnti-anti-idiotypic withAssay (Ab3) generated Id MoAb M7-625 antisera had the specific reactivity to CEA, since absorbed antisera with purified CEA completely failed to (Ab2)A. show M, 200,000 band (CEA) in Western blot analysis (data T3-503T4-202M7-049M7-625B.Sandwich assay not shown). These results suggest that anti-anti-idiotypic MoAb M7-625 antiserum (Ab3) may have an antibody population of the same specificity as anti-CEA MoAb MA208 (Abl). On the other Inhibition assay T3-503T4-202M7-049M7-625T3-50331,708"1.0367931.22397*2(11T4-2024665,09550592108542M7-0496141.2625,9221,74330970M7-6255651,12273913,78200096 hand, detailed results are not shown here, but 4 MoAbs (0.2%), including M7-625, are anti-idiotypic antibodies ÃŸ(Ab2/3),and 28 MoAbs (1.5%), including M7-049, are anti-idiotypic anti " Anti-anti-idiotypic antisera were incubated with anti-idiotypic monoclonal antibodies coated on microtiter plates, and then they were tested for the reactivity bodies y (Ab27), when hybridomas were screened with sand "t ' ' I l.il i-k'il anti-idiotypic monoclonal antibodies in sandwich assay. The results wich assay and inhibition assay. are expressed as bound cpm in sandwich assay (A). * Anti-anti-idiotypic MoAb T3-503. T4-202, M7-049. and M7-625 antisera were preincubated with '"l-labclcd MoAb T3-503, T4-202, M7-049, and M7- DISCUSSION 625, and then the mixtures were added to microtiter plates coated with MoAb 5B3 or MA208. After being washed, the bound radioactivity was counted. The Anti-idiotypic antibodies are of interest from the viewpoint results are expressed as percentage of inhibition, compared with that of preim mune serum as a control in inhibition assay (B). of new immunomodulators for the treatment of cancer patients. Administration of anti-Id antibodies might trigger an anti- 5B3, and M7-049 and M7-625 to MoAb MA208 in the synge- tumor humoral and/or cellular immune response in hosts with neic system was done to examine whether they could induce cancer. The mechanism involved in treatment with anti-Id anti-anti-Id antibodies. This immunization was repeated 7 times antibodies is quite different from that with conventional anti- for anti-Id MoAbs T3-503 and T4-202, and 5 times for M7- tumor-associated antigen antibodies. Anti-tumor-associated an 049 and M7-625. Among antisera obtained, the strongest sera tigen antibodies directly bind to cancer cells and then show in terms of sandwich assay were selected and used for inhibition cytotoxic and/or inhibitory effects, while anti-Id antibodies are assay. The results of this study, summarized in Table 4, Section meant to induce the immune response of the host which could A, show that anti-Id MoAbs (Ab2) could induce anti-anti-Id be beneficial in its anti-tumor effects. antibodies (Ab3) specifically reactive to the corresponding im The results reported here demonstrate several aspects of the munizing anti-Id MoAb, but not to irrelevant MoAbs in sand idiotype network system in carbohydrate or peptide moiety of wich assay. The induction of anti-anti-Id antibodies specific to CEA, which may occur in vivo when anti-Id MoAb is injected. corresponding immunizing anti-Id MoAb was also suggested, Four distinct idiotopes, recognized with anti-Id MoAb T3-503, since anti-Id MoAb-immunized antisera (Ab3) showed inhibi T4-202, M7-049, and M7-625, could induce anti-anti-Id anti tory activity to the binding of anti-idiotype MoAb (Ab2) to bodies (Ab3) specific to the corresponding idiotope at the anti-CEA MoAb 5B3 or MA208 (Abl) (Table 4, Section B). combining site of anti-CEA MoAb 5B3 and MoAb MA208 in Reactivities of Anti-Anti-Idiotypic Antibodies to CEA. In order a syngeneic system. The anti-anti-Id antibodies that were in to investigate the internal image of antigen (CEA), the reactiv duced only with anti-Id MoAb M7-625 were found to have the ities of four anti-anti-idiotypic antisera to CEA were tested in same reactivity as that of MoAb MA208, whereas the anti-anti- competition assay and in immunoblotting assay. As shown in Id antibodies induced with other anti-Id MoAbs were not. It Fig. 2, anti-anti-idiotypic MoAb M7-625 antiserum competed might suggest, therefore, that anti-Id MoAb M7-625 bears the with i:'I-labeled MoAb MA208 to CEA (Fig. 2B), while anti- internal image of the antigen, CEA, the chemical nature of anti-idiotypic MoAb M7-049 antiserum, T3-503 antiserum, which appears to be a peptide. It has been already confirmed and T4-202 antiserum did not compete with either I25l-labeled that there exists the homology of amino acid sequences between MoAb 5B3 (Fig. 2A) or 125I-labeled MoAb MA208 (Fig. 2B) anti-Id MoAb M7-625 and CEA, according to the cloning to purified CEA. Furthermore, immunoblotting assay showed results of complementary determining regions 1, 2, and 3 of

(A) (B) 80 80

60 60

Fig. 2. Competitive activity of anti-anti-idi 40 40 otypic MoAb T3-503 (D). T4-202 (â€¢),M7-049 (O), and M7-625 (â€¢)antisera (Ab3) with '"1- labcled MoAb 5B3 (A) or MA208 (B) (Abl), which binds to purified CEA. 20 20-

1:10 1:50 1:250 1:10 1:50 1:250

Serial dilution of anti-anti-Id antisera

2602

1 2 3

(Kd) 200- f

116- 97- Fig. 3. Western blot analysis of binding activ ities of anti-anti-idiotypic MoAbs T3-503 (Lane 2), T4-202 (Lane 3), M7-049 (Lane 5). and M7- 625 (Lane 6) antisera to purified CEA. Lane I 66- (anti-CEA MoAb 5B3). Lane4 (anti-CEA MoAb MA208), and Lane 7 (preimmune serum) are tested for binding to CEA as controls.

43-

Anti-CEA Anti-MoAb Anti-MoAb Anti-CEA Anti-MoAb Anti-MoAb Preimmune MoAb 5B3 T3-503 T4-202 MoAb M7-049 M7-625 serum antiserum antiserum MA208 antiserum antiserum

MoAb M7-625.5 This result suggests that only a restricted patients have shown partial clinical remission (33). They have idiotope at the antigen-combining site of Ab2 may mimic the also found that the anti-Id antibodies might carry the internal structure of the epitope of the antigen and have the internal image of the antigen in this particular tumor-associated antigen image of the epitope. It does not necessarily mean, however, system, since xenogeneic anti-Id antibodies (Ab2) made it pos that an anti-Id MoAb to a MoAb which was raised against an sible to develop antigen-specific anti-anti-Id MoAbs (Ab3) (34). epitope of the peptide has an advantage over that raised against On the other hand, several reports (35-37) have indicated an epitope of the carbohydrate of CEA in inducing anti-anti-Id the induction of cellular immunity specific to tumor-associated antibodies which react with the antigen. Some kinds of anti-Id antigens by immunization of anti-Id antibodies in the experi MoAbs also had the internal image of antigen in carbohydrate mental system. Lee et al. (35) showed a delayed type hypersen- antigen system, because anti-Id MoAbs to anti-CEA or anti- sitivity reaction specific to a murine oncofetal bladder tumor MAA MoAbs that recognized distinct carbohydrate moiety of antigen with use of a syngeneic anti-Id MoAb. Furthermore, it CEA or MAA had already been established, and were able to is interesting that an anti-Id MoAb could activate the tumor- induce anti-anti-Id antibodies which had reactivities to the specific cytotoxic T-lymphocytes in murine lymphoma, as de antigens (data not shown). The study presented here focuses scribed by Raychaudhuri et al. (36, 37). These studies might mainly on induction of humoral immunity with anti-Id MoAbs indicate that one possible strategy for the generation of tumor in a syngeneic system. It is well known that human antibodies immunity (not only humoral but also cellular) is vaccination or derived from a host's own system are more effective for anti immunization of cancer patients with anti-Id antibodies that body-dependent cell-mediated cytotoxicity than xenogeneic an mimic the tumor antigen structure. This type of study (in vira tibodies (28). In addition, there is growing evidence that im- experiments) is now under investigation in our laboratory, using munoglobulin idiotypes influence the repertoires of both helper syngeneic murine melanoma cells and anti-Id MoAbs to anti- and suppressor T-cells (29, 30). In our preliminary experiments, GM, ganglioside MoAb, collaborating with Dr. Masaru Tani- anti-anti-Id antibodies induced in nu/nu mice with anti-Id guchi of Chiba University. We believe that this approach, using MoAb M7-625, reacted with purified CEA. These humoral anti-Id antibody to modulate the idiotype network system, immune responses, however, could not suppress the CEA- would be a new immunotherapy for cancer. producing tumor progression in in vivo experiments (data not shown). Investigation of T-cells is absolutely necessary for ACKNOWLEDGMENTS further analysis of the idiotype network concerning tumor- associated antigens on carcinoma cells. We acknowledge the technical help of M. Takeda and the secretarial Koprowski et al. (31) have reported the possibility of a assistance of H. Yamashita. beneficial role of anti-Id antibodies induced in patients after administration with a MoAb, designated as CO 17-1A, to colo- REFERENCES rectal tumors. They have recently demonstrated that cancer patients treated with this MoAb (Abl) developed not only anti- 1. Jerne, N. K. Towards a network theory of the immune system. Ann. Irnniu- Id antibodies (Ab2), but also anti-anti-Id antibodies (Ab3) to nol., 125: 373-389, 1974. 2. Bhattacharya-Chatterjee, M.. Chatterjee. S. K.. Vasile, S.. Scon. B. K., and their autologous Ab2 (32). Their results indicate that idiotype KÃ¶hler.H. Idiotype vaccines against human T cell lymphoblastic leukemia. cascades might be activated by the Abl injection and the anti II. Generation and characterization of a monoclonal idiotype cascade (Abl. Ab2 and Ab3). J. Immunol.. 141: 1398-1403. 1988. gen-specific Ab3 responses might underlie delayed clinical 3. Raychaudhuri, S., Saeki. Y., Chen. J-J.. Iribc, H., Fuji, H.. and KÃ¶hler.H. course which is often observed in cancer patients treated with Analysis of the idiotypic network in tumor immunity. J. Immunol., 139: MoAbs. Furthermore, they have tried to treat 13 cancer patients 3902-3910, 1987. 4. Cleveland, W. L., Wassermann, N. H., and Sarangarajan. R. Monoclonal with polyclonal anti-Id antibodies (Ab2), with the result that 6 antibodies to the acctylcholinc receptor by a normally functioning auto-anti- idiotypic mechanism. Nature (Lond.), 305: 56-57, 1983. *Submitted for publication. 5. Mendlovic. S.. Brocke. S.. Shoenfeld. Y.. Ben-Bassat, M., Meshorer. A.. 2603

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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1991 American Association for Cancer Research. Induction of Antigen-specific Immune Response with Use of Anti-Idiotypic Monoclonal Antibodies to Anti-Carcinoembryonic Antigen Antibodies

Masayuki Tsujisaki, Kohzoh Imai, Shigeru Tokuchi, et al.

Cancer Res 1991;51:2599-2604.

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