Original Paper Tumor Biol 1992;13:226-236
Yoshiaki Hanzawa3’b Masayuki Tsujisaki3 Shigeru Tokuchia Takeshi Moriyamab Masaaki Izawab Kohzoh Im ai3 Akira Yachia
Detection of Xenogeneic Anti-Idiotypic Antibodies Specific to Murine Monoclonal Antibody 17-1A in Patients with Gastrointestinal Cancer
Department of Internal Medicine (Section 1), Sapporo Medical College, Sapporo; Division of Drug Development, Ajinomoto Central Research Laboratories, Yokohama, Japan
Abstract
Murine monoclonal antibody (MoAb) 17-1 A, which reacts with an adenocarcinoma-associated antigen, has recently been utilized in a phase I clinical trial for patients with gastrointesti nal tract cancers in Japan. In order to analyze anti-idiotypic (Id) antibodies to murine MoAb 17-1A in the sera of these cancer patients, we established a simple and specific assay. In a modified sandwich assay, normal mouse sera were utilized for neutralization of rat antimouse immunoglobulin (Ig) anti bodies and reduction of their nonspecific effects. When the modified sandwich assay was applied to the sera of patients who had been treated with MoAb 17-1 A, anti-id antibodies were induced in 53% of 13 cancer patients with gastrointesti nal cancers at 3-4 weeks after infusion of MoAb.
Introduction
Murine monoclonal antibody (MoAb) 171A (IgG2a), which reacts with a 37-kD glyco protein on colorectal carcinoma cells, has been characterized and used as an immunotherapeutic agent for gastrointestinal cancers [1-7]. Recently, a phase I clinical trial of
Received: November II, 1991 Accepted: May 1 ,1992
MoAb 17-1A has also been carried out in Japan. It has been reported that one of the mechanisms of antitumor activity induced with MoAb 17-1A is an antibody-dependent cell-mediated cytotoxicity [8, 9], It is of inter est that Koprowski et al. [4] have suggested the possible beneficial role of anti-idiotypic (Id) antibodies induced in cancer patients ad-
K. Imai Department of Internal Medicine (Section 1) Sapporo Medical College SI W 16Chuo-ku Sapporo 060 (Japan)
© 1992 S. Karger AG, Basel 1010-4283/92/ 0134-022652.75/0
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Keyw ords Anti-idiotypic antibody Immunotherapy with monoclonal antibody Murine monoclonal antibody 17-1A
Materials and Methods MoAb and Antisera for Assays MoAb 17-1A to adenocarcinoma-associated anti gens on gastrointestinal cancer cells was previously described [2] and is manufactured by Centocor Inc. (Malvern, Pa., USA) for clinical application to cancer patients. Goat antirat Ig antisera and goat antihuman Ig antisera for assays, Cappel No. 0213-0231 and 0601-0231, respectively, were purchased from Orga non Teknika (West Chester, Pa., USA). Murine anti-id MoAb M7-049 against anti-CEA MoAb MA208 was developed as described elsewhere [14],
Purification and Radiolabelling MoAbs were purified from ascitic fluid either by affinity chromatography on protein A-Sepharose [15], by ion-exchange chromatography on DEAE or by caprylic acid precipitation [ 16]. Antibodies were radiolabelled with 125I, utilizing the chloramine T method [17]. Development o f Anti-Id and Antimouse Ig Antibodies Wistar rats were primed with 100 pg of purified murine MoAb 17-1A coupled to keyhole limpet hemocyanin (KLH) and polymerized with glutaraldehyde in complete Freund’s adjuvant (Gibco Laboratories, De troit. Mich., USA) [ 18]. On the 7tn and 14th day, the rats were boosted with the same immunogen in incomplete Freund’s adju vant. Three days after the last booster, rat sera were obtained and utilized for the detection of antimouse Ig antibodies and anti-id antibodies. Detection o f Anti-MoAb 17-1A Antibodies Ninety-six-well polyvinyl chloride microtiter plates (Dynatech, Alexandria, Va., USA) were coated with MoAb 17-1A by incubating 100 pi of a MoAb solution (100 pg/ml) in 0.1 M bicarbonate buffer, pH 9.5, for 16 h at 4°C. Following 3 washings with phosphatebuffered saline (PBS), pH 7.4, containing 0.05% Tween 20 (PBS-T20), the plates were blocked with 5 % bovine serum albumin (BSA) for 4 h. After washing, 100 pi of the diluted rat or human sera was added to the plates and incubated for 3 h. Following 3 washings, 125I-labelled goat antirat Ig (1 X 105 cpm) or goat anti human Ig (1 X 105 cpm) was incubated for 3 h at 4 °C. The plates were then washed 5 times with PBS-T20, the wells were cut and the radioactivity was counted in a gamma-counter. The assay was done in triplicate and the mean values were presented. Detection o f Anti-Id Antibodies Inhibition Assay. This assay was performed as de scribed [19], Fifty microliters of 125I-labelled MoAb 17-1A (1 X 105 cpm) was incubated for 2 h at 4 °C with 50 pi of diluted rat serum. Then, the mixture was added to 17-lA-bearing colonic carcinoma SW1116 cells (1 X lOVwell). At the end of another 2 h of incu bation at 4 ° C, the cells were washed 5 times with PBS, and the bound radioactivity was counted in a gammacounter. Results were expressed as the percentage of binding inhibition and were compared with the con trol serum which had been immunized with the irrele vant MoAb or with preimmune serum.
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ministered MoAb 17-1A as another mecha nism for an antitumor effect [4, 10, 11], Inves tigations have been undertaken to ascertain whether the idiotype network system has an important role to play in the regulation of immune responses [ 12, 13], as such responses may eventually result in the destruction of tumor cells. Therefore, it is important to mea sure the levels of anti-id antibodies as well as antimouse immunoglobulin (Ig) antibodies and to analyze the mechanism of possible immunotherapeutic effects on these patients. However, there have been some difficulties in detecting anti-id antibodies in the sera of patients who have been injected with murine MoAb, since antimouse Ig antibodies, as well as anti-id antibodies, may simultaneously be induced by and affect the assays. Herlyn et al. [ 10] have presented a specific assay system for the detection of anti-id anti bodies in patient sera. However, the proce dure to prepare the purified goat anti-id anti bodies for the assay requires many steps. In the present study, we established a simpler assay for the detection of anti-id anti bodies in a xenogeneic model system and investigated the incidence and kinetics of in duction of anti-id antibodies and antimouse Ig antibodies in cancer patients administered murine MoAb 17-1 A.
Clinical Studies Serum from 13 metastatic or recurrent cancer (8 gastric and 5 colonic cancer) patients was used in this study. All patients were administered purified murine MoAb 17-1A in the phase I trial in Japan (Chairman: Prof. T. Taguchi, Osaka University School of Medi cine). These patients were injected systemically with one dose of MoAb (25, 100 or 1,000 mg) in a single infusion. The patient sera for detection of antimouse Ig or anti-ld antibodies were obtained at 1-week inter vals after the administration of MoAb 17-1 A. The sandwich assay and the modified sandwich assay de scribed above were used in order to detect antimouse Ig and anti-id antibodies, respectively.
Results
Detection o f Antimouse Ig Antibodies in Rat Serum Elicited with Murine MoAbs In order to detect antimouse Ig antibodies in a xenogeneic system, as a model for human patients who were injected with murine MoAb, rats were immunized with murine MoAbs. Serially diluted sera from rats immu nized with purified murine MoAb 17-1A (IgG2a) or irrelevant murine MoAb M7-049 (IgG2a) were tested in the binding assay with the use of coated MoAb 17-1A as a catcher and 125I-labelled goat antirat Ig antibodies as a tracer. As shown in figure 1, not only the rat sera immunized with MoAb 17-1A but also those immunized with MoAb M7-049 reacted
228
remarkably with MoAb 17-1 A, whereas preimmune rat sera did not. Detection o f Anti-Id Antibodies in Immunized Rat Sera Two different types of assay were estab lished to detect anti-id antibodies specific to MoAb 17-1A in this xenogeneic system. The first was the inhibition assay, which demonstrates the inhibitory activity of rat sera against the binding of l25I-labelled MoAb 17-1A to antigen. Human colonic carcinoma SW1116 cells, which express 17-1A antigen, were used as targets in this assay. The serum of rats who had been immunized with MoAb 17-1A remarkably inhibited the binding of l25I-labelled MoAb 17-1A to the cells, as shown in figure 2, while the serum of rats who had been immunized with MoAb M7-049 failed to inhibit. This inhibition, however, was affected when MoAb 17-1A was added to the serum (data not shown). More than 10 pg/ ml of MoAb 17-1A in the serum blocked the binding inhibition, suggesting that this assay should not be utilized when a concentration of MoAb 17-1A is higher than 10 pg/ml in the serum. The second system to detect anti-id anti bodies is the sandwich assay with use of MoAb 17-1 A. Serum of rats who had been immunized with murine MoAb 17-1A was applied to this assay. The binding activities of rat anti-MoAb 17-1A and anti-MoAb M7-049 seru m to MoAb 17-1A were detected in a con centration-dependent manner, while that of preimmune rat serum was not, as shown in figure 3a. This reaction can be explained as follows: the antimouse Ig antibodies in antiMoAb 17-1A serum and also in anti-MoAb M7-049 serum, which were induced simulta neously in rats, reacted with mouse MoAb 171A, which was used as a catcher and a tracer. There is a possibility, however, that the antiid antibodies to MoAb 17-1A were induced.
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Sandwich Assay. As described elsewhere [18], a sandwich assay was performed. Briefly, MoAb-17-lAcoated microtiter plates were blocked with 5 % BSA for 4 h, and then 100 pi of diluted rat serum was added and incubated for 4 h. Following 3 washings, the plates were incubated with l25l-labclled MoAb I7-1A for4 h. The plates were then washed 5 times and the radioac tivity was counted. On the other hand, a modified sandw'ich assay was also done, utilizing 50 pi of 1:10-diluted normal mouse serum which was preincubated with 50 pi of diluted rat or human serum before being applied to MoAb-17-1Acoatcd plates. Both assays were done in triplicate, and the mean values were presented.
1:40
1:160 1:80
1:320
1:640 1:2,560 1:10.240 1:1,280 1:5,120
Serial dilution of rat sera
Dilution of rat sera
Fig. 1. Detection of antimouse Ig antibodies against murine MoAb 17-1A in sera of rats immunized with murine MoAb 17-1A. Rat anti-MoAb 17-1A serum (o), anti-MoAb M7-049 serum (A ) and preimmune serum (□) were serially diluted with PBS and tested, utilizing coated MoAb 17-1A as a catcher and l25l-labelled antirat Ig antibodies as a tracer. Fig. 2. Inhibitory activities of diluted rat antiMoAb 17-1A serum (o), anti-MoAb M7-049 serum (A ) and preimmune serum (□) on l25I-labelled MoAb 17-1A binding to colonic carcinoma SW 1116 cells.
Fig. 3. Sandwich assay for the detection of anti-id antibodies to MoAb 17-1 A. a Serially diluted rat antiMoAb 17-1A serum (o), anti-MoAb M7-049 serum ( a ) and preimmune serum (□) were incubated with MoAb-17-IA-coated microtiter plates and then tested for reactivity of l25l -labelled MoAb 17-1 A. b The mix ture of serially diluted rat anti-MoAb 17-1A serum (o), anti-MoAb M7-049 serum (A ) or preimmune serum (□), and 1:10-diluted normal mouse serum was tested in a modified sandwich assay.
In order to reduce the effect of coinduced antimouse Ig (non-anti-Id) antibodies in the rat, the excess normal mouse serum was incu bated with rat sera before application of these sera to the sandwich assay. Figure 3b shows the results of the sandwich assay using the mixture of MoAb immunizing rat sera and
normal mouse sera to neutralize the activity of the anti mouse Ig antibodies. The reactivity of MoAb M7-049 immuniz ing rat serum was completely reduced by the addition of normal mouse serum, although that of MoAb 17-1A immunizing rat serum retained the reactivity. The relative percent
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3a
age of antimouse activity which was due to anti-id would be approximately 60% for MoAb 17-1A and 9% for MoAb M7-049. A sandwich assay using MoAb M7-049 as a catcher and a tracer was also performed to measure the anti-ld antibodies to MoAb M7049. It showed a similar tendency (fig. 4a.b) to that of MoAb 17-1 A, suggesting that this modified sandwich assay is useful for detect ing anti-id antibodies. Anti-constant region antibodies in rat serum have been mainly responsible for disturbing sandwich assays to detect anti-id antibodies. In order to deter mine the dose (or volume) of normal mouse serum to be preincubated with rat sera, the neutralizing activities of normal mouse se rum were tested in a sandwich assay. As shown in figure 5, a 1:50 dilution of normal mouse serum was sufficient to neutralize the antimouse Ig antibodies which were induced in 1:10-diluted rat sera. Addition of normal mouse serum even reduced the binding of 17-1A up to approximately 15% (26,000 cpm, at 1:5.000 dilution, to 22,000 cpm, at 1:50 dilution, bound). The presence of MoAb 17-1A in the rat serum is unlikely to affect this sandwich assay, since the concen tration of MoAb 17-1A in the serum should be too low.
Fig. 4. Sandwich assay for detection of anti-ld anti bodies to MoAb M7-049. Immunoassay was per formed as described in figure 3 using MoAb M7-049 as the capture and detection antibody, a, b Rat antiMoAb 17-1A serum (o), anti-MoAb M7-049 scrum (A) and preimmune serum (□).
Kinetics o f the Anti-Id Antibodies and Antimouse Ig Antibodies Induced with MoAb 17-1A The rats immunized with KLH-conjugated MoAb 17-1A were bled on days 7, 21, 42, 49 and 60 after the immunization, and the ob tained sera were used to measure both anti-id antibodies and antimouse Ig antibodies. The anti-ld antibodies to MoAb 17-1A were grad ually induced in rats and continued to be gen erated even after 60 days (fig. 6). On the other hand, the antimouse Ig antibodies were rap idly induced after the immunization and kept a high titer.
Detection o f Anti-Id Antibodies and Antimouse Antibodies in Patient Sera Thirteen patients with gastrointestinal ad enocarcinoma were injected with a purified sterile pyrogen-free preparation of MoAb 171A by a single infusion. Four of the 13 pa tients received 1,000 mg, 4 patients 100 mg and 5 patients 25 mg of MoAb 17-1 A. As shown in figure 7, 7 of the 13 patients who received the MoAb injection developed anti-ld antibodies to MoAb 17-1 A; 5 patients (cases 3, 4, 5, 11 and 12) showed a peak level
Dilution of rat sera
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Dilution of rat sera
Fig. 5. The effect of normal mouse serum on neu tralizing the rat antimouse Ig antibodies in a modified sandwich assay. Serially diluted normal mouse sera were preincubated with 1:10-diluted rat anti-MoAb 17-1A serum (o), anti-MoAb M7-049 serum (A) and preimmunc scrum (□).
Fig. 6. Changes in the levels of anti-id antibodies (o) and antimouse Ig antibodies (•) in the serum of rats
immunized with MoAb 17-1 A. Rat sera (1:10 and 1:100 dilution) were used to detect anti-id antibodies in a modified sandwich assay and antimouse Ig anti bodies in a binding assay (coated MoAb 17-1A and l25I-label!ed antirat Ig antibodies).
Table 1. Induction of anti-MoAb 17-1A antibodies and anti-id antibodies in the sera of patients by adminis tration of murine MoAb 17-1A
Induction of antibodies in patients
MoAb 17-1A injected 1,000 mg
100 mg
25 mg
Anti-MoAb 17-1A antibodies
4/4
100%
4/4
100%
4/5
80%
12/13
92%
Anti-Id antibodies
2/4
50%
3/4
75%
2/5
40%
7/13
54%
1A antibodies including both anti-constant region and anti-id antibodies, and their levels in sera changed in a pattern similar to those of anti-id antibodies. The highest level in the above 5 patients (cases 3, 4, 5, 11 and 12) was detected 2-3 weeks after the injection, and the level had no direct relation to the dose of MoAb 17-1A (fig. 7). The summary of the induction of antibodies in patients is shown in table 1.
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of anti-id antibodies 2-3 weeks following the administration of MoAb, whereas the other 2 patients (cases 6 and 8) at 6-8 weeks. Both cases 9 and 13 appear to have developed negligible antimouse Ig and anti-id. The induction levels of anti-id antibodies were unrelated to the administered doses of MoAb 17-1 A. With regard to human anti mouse Ig antibodies in patient sera, 12 of the 13 patients developed human anti-MoAb 17-
total
30.000
4,000
Case 1
20.000
30,000
Case 1
3,000
10,000 •
4,000
Case 5
20,000 | "
2,000
Case 5
3,000
P /v v .
10,000
2,000 /* •• •
0
1,000 0
4
8
12
0
4
8
12
0
4
8
12
1,000
0 0
4
8
12
0
4
8
12
a
b
232
modified sandwich assay were used for the detection of respective antibodies, a 4 cases injected with 1,000 mg MoAb. b 4 cases injected with 100 mg MoAb. c 5 cases injected with 25 mg MoAb.
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Fig. 7. Detection of human anti-MoAb 17-1A anti bodies (•. left panels) and anti-ld antibodies (o, right panels) in the sera of 13 patients who received murine MoAb 17-1A by a single infusion. Sandwich assay and
30.000
cpm
20.000 10,000
0 0
4
8
12
0
4
8
12
0
4
8
12
0
4
8
12
30.000
cpm
20.000 10,000
.....
cpm
cpm
0
30.000
4.000
Case 13
20.000
3.000
10.000 . \
2.000
Case 13
«
w*
1.000 4
8
Weeks
c
12
D
4
8
Weeks
12
Although several assays to detect anti-id antibodies generated in a xenogeneic system have been tried, they have not been feasible for a routine test since the specificity of the assays was not satisfactory and/or the assays were complicated. Herlyn et al. [10] reported on assays to detect anti-id antibodies reacting with combining and non-combining site-re lated idiotypes in murine MoAb 17-1 A. In that assay, it was necessary to prepare poly clonal anti-id antibodies to MoAb 17-1A which were isolated from animal sera (rabbit or goat) following absorption of the sera with the use of immunoabsorbent columns which were prepared with idiotypically unrelated MoAb of the same isotype (IgG2a). We tried to apply two different types of assays, one an inhibition assay and the other a sandwich assay, for the detection of xeno geneic anti-id antibodies. The inhibition as say demonstrates the inhibitory activity of rat serum generated with murine MoAb 17-1A on the binding o f 125I-labelled MoAb 17-1A to antigen-bearing cells. As shown in figure 2, MoAb 17-1A immunizing rat serum inhibited 125l-labcllcd MoAb 17-1A binding to the cells, whereas neither preimmune rat serum nor MoAb M7-049 immunizing rat serum were inhibitory. These results suggest that the in hibitory activity is not affected by antimouse Ig antibodies. The sandwich assay, which was established to detect anti-id antibodies in a syngeneic sys tem as described elsewhere [ 18], was applied to detect xenogeneic anti-ld antibodies. Both rat anti-MoAb 17-1A and anti-MoAb M7-049 sera reacted remarkably with murine MoAb 17-1A in the unmodified sandwich assay (fig. 3a). suggesting that not only anti-id anti bodies specific to MoAb 17-1A but also anti mouse Ig antibodies were detected in this assay.
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Discussion
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ually decreased (data not shown). These data are similar to those reported by Herlvn et al. [Hi-
Immune responses in cancer patients fol lowing a single infusion of MoAb 17-1A were investigated. As a result, the induction level of anti-id and antimouse Ig antibodies and dura tion time were found to be unrelated to the injection doses (25, 100 or 1,000 mg) of MoAb 17-1A (fig. 7a-c, table 1). Fifty-four and 92% of the patients developed anti-id and antimouse Ig antibodies, respectively. The incidence of positivity for induction of anti-id antibodies is almost the same as that in the phase II study reported by Sears et al. [20], The levels of anti-Id and antimouse Ig antibodies simultaneously fluctuated and their levels in most of the cases maximized 23 weeks after the injection. A pharmacoki netic study by Khazaeli et al. [3] has shown that most patients had an antibody response with peak levels occurring 15-20 days after infusion in the phase I trial. With regard to the differences between anti-Id antibody in duction noted in rat sera and that of patient sera, it is speculated that rats immunized with intraperitoneal injection of KLH-conjugated MoAb in Freund’s adjuvant had a continuous induction of anti-Id antibodies, while patients 661 blistered a single infusion of MoAb showed a rapid increase and reduction in antiId antibodies in the sera. Hcrlyn et al. [ 11 ] have indicated that a sin gle injection of MoAb to a cancer patient could continue inducing anti-Id antibodies (but not antimouse Ig antibodies) for 400 days after administration. Our studies [21, 22] have also suggested that syngeneic anti-Id an tibodies could be detected for more than 200 days after intraperitoneal injection of KLHconjugated MoAb. The reason why contin uous production of anti-Id antibodies was maintained was unclear at the time of this study. This phenomenon, however, may be
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The addition of normal mouse sera to im munized rat sera and their preincubation are of use in order to eliminate the influence of rat antimouse Ig antibodies. As shown in fig ure 3b, rat anti-id antibodies to murine MoAb 17-1A could be specifically detected in the modified sandwich assay, using the excess of normal mouse serum to neutralize antimouse Ig antibody activities. When rat anti-MoAb M7-049 serum was tested, antiid antibodies specific to MoAb M7-049 were detected in the modified sandwich assay us ing MoAb M 7-049 (fig. 4b) but were not spe cifically detected in the unmodified proce dure (fig. 4a). These results suggest that the modified sandwich assay is of use to detect anti-id antibodies specific to the immunizing MoAb. Furthermore, it is suggested that it was necessary to add a 1:50 dilution of normal mouse serum to rat sera diluted 1:10 in order to reduce the effects of anti mouse Ig and other antibodies (fig. 5). Normal mouse sera showed more effective neutralizing ability than an unrelated murine MoAb (IgG2a; data not shown), since they contain a panel of murine antibodies with different isotypes and different amino acid sequences in the frame work of the variable regions. Using the modified sandwich assay to de tect anti-id antibodies and the binding assay with coated MoAb 17-1A and l25I-labelled antirat Igs to detect antimouse Ig antibodies, the time course of anti-id and antimouse Ig antibody production was investigated in rats which were immunized 3 times with murine MoAb 17-1A at 0, 7 and 14 days. Figure 6 shows that the anti-id antibodies were gener ated gradually and continued to be produced for up to 60 days and that antimouse anti bodies were rapidly induced after the first infusion of MoAb. The high level of anti-id antibodies in rat sera was maintained after 60 days, while the anti mouse antibodies grad
important in the hosts’ immune response against tumor progression. Although possible correlations between the clinical status of the patients and their im mune response to the mouse monoclonal anti body were not clear at the time of this study, they should be analyzed in the near future. From the viewpoint of the idiotype network theory, internal-image-bcaring anti-id anti bodies may be of use for immunotherapy to induce an antitumor immune response as an antigen-specific immunomodulator. Based on this strategy, we have developed and analyzed the internal-image-bearing anti-id MoAbs which are related to carcinocmbryonic anti gen [14], Further studies are now under way in our laboratory to clarify the relationship between the induction of anti-id antibodies
and the prognosis of cancer patients and the contribution of cellular immunity as well as humoral immunity.
Acknowledgements This work was supported by a Grant-in-Aid for Co operative Research (AY, KI), by a Grant-in-Aid for Scientific Research on Priority Areas (KI) and by a Grant-in-Aid for Cancer Research (MT, KI, AY) from the Ministry of Education, Science and Culture, and was also supported by Grants for Cancer Research (AY) from the Ministry of Health and Welfare, Japan. The technical assistance of Ms. M. Nosaka, Ms. K. Kawamata and Ms. M. Ohe is greatly appreciated. We especially thank Dr. Y. Hinoda and Dr. T. Sugiyama for helpful advice, Mr. T. Mimura for grateful support and Ms. H. Yamashita for secretarial assistance.
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13 Powell TJ, Spann R, Nguyenduc M, Lamon EW: Induction of effective immunity to moloney murine sar coma virus using monoclonal antiidiotypic antibody as immunogen. J Immunol 1989; 142:1318-1324. 14 Tsujisaki M, Imai K, Tokuchi S, Hanzawa Y, Ishida T. Kitagawa H, Yachi A: Induction of antigen-spe cific immune response with use of anti-idiotypic monoclonal anti bodies to anti-carcinoembryonic an tigen antibodies. Cancer Res 1991; 51:2599-2604. 15 Ey L, Prowes J, Jenkin R: Isolation of pure IgG 1, lgG2a and IgG2b im munoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 1978;15:429-436. 16 Russo C, Callegaro L, Lanza E, Ferrone S: Purification of IgG mono clonal antibody by caprylic acid pre cipitation. J Immunol Methods 1983;65:269-271.
Yoshiaki Hanzawa3’b Masayuki Tsujisaki3 Shigeru Tokuchia Takeshi Moriyamab Masaaki Izawab Kohzoh Im ai3 Akira Yachia
Detection of Xenogeneic Anti-Idiotypic Antibodies Specific to Murine Monoclonal Antibody 17-1A in Patients with Gastrointestinal Cancer
Department of Internal Medicine (Section 1), Sapporo Medical College, Sapporo; Division of Drug Development, Ajinomoto Central Research Laboratories, Yokohama, Japan
Abstract
Murine monoclonal antibody (MoAb) 17-1 A, which reacts with an adenocarcinoma-associated antigen, has recently been utilized in a phase I clinical trial for patients with gastrointesti nal tract cancers in Japan. In order to analyze anti-idiotypic (Id) antibodies to murine MoAb 17-1A in the sera of these cancer patients, we established a simple and specific assay. In a modified sandwich assay, normal mouse sera were utilized for neutralization of rat antimouse immunoglobulin (Ig) anti bodies and reduction of their nonspecific effects. When the modified sandwich assay was applied to the sera of patients who had been treated with MoAb 17-1 A, anti-id antibodies were induced in 53% of 13 cancer patients with gastrointesti nal cancers at 3-4 weeks after infusion of MoAb.
Introduction
Murine monoclonal antibody (MoAb) 171A (IgG2a), which reacts with a 37-kD glyco protein on colorectal carcinoma cells, has been characterized and used as an immunotherapeutic agent for gastrointestinal cancers [1-7]. Recently, a phase I clinical trial of
Received: November II, 1991 Accepted: May 1 ,1992
MoAb 17-1A has also been carried out in Japan. It has been reported that one of the mechanisms of antitumor activity induced with MoAb 17-1A is an antibody-dependent cell-mediated cytotoxicity [8, 9], It is of inter est that Koprowski et al. [4] have suggested the possible beneficial role of anti-idiotypic (Id) antibodies induced in cancer patients ad-
K. Imai Department of Internal Medicine (Section 1) Sapporo Medical College SI W 16Chuo-ku Sapporo 060 (Japan)
© 1992 S. Karger AG, Basel 1010-4283/92/ 0134-022652.75/0
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Keyw ords Anti-idiotypic antibody Immunotherapy with monoclonal antibody Murine monoclonal antibody 17-1A
Materials and Methods MoAb and Antisera for Assays MoAb 17-1A to adenocarcinoma-associated anti gens on gastrointestinal cancer cells was previously described [2] and is manufactured by Centocor Inc. (Malvern, Pa., USA) for clinical application to cancer patients. Goat antirat Ig antisera and goat antihuman Ig antisera for assays, Cappel No. 0213-0231 and 0601-0231, respectively, were purchased from Orga non Teknika (West Chester, Pa., USA). Murine anti-id MoAb M7-049 against anti-CEA MoAb MA208 was developed as described elsewhere [14],
Purification and Radiolabelling MoAbs were purified from ascitic fluid either by affinity chromatography on protein A-Sepharose [15], by ion-exchange chromatography on DEAE or by caprylic acid precipitation [ 16]. Antibodies were radiolabelled with 125I, utilizing the chloramine T method [17]. Development o f Anti-Id and Antimouse Ig Antibodies Wistar rats were primed with 100 pg of purified murine MoAb 17-1A coupled to keyhole limpet hemocyanin (KLH) and polymerized with glutaraldehyde in complete Freund’s adjuvant (Gibco Laboratories, De troit. Mich., USA) [ 18]. On the 7tn and 14th day, the rats were boosted with the same immunogen in incomplete Freund’s adju vant. Three days after the last booster, rat sera were obtained and utilized for the detection of antimouse Ig antibodies and anti-id antibodies. Detection o f Anti-MoAb 17-1A Antibodies Ninety-six-well polyvinyl chloride microtiter plates (Dynatech, Alexandria, Va., USA) were coated with MoAb 17-1A by incubating 100 pi of a MoAb solution (100 pg/ml) in 0.1 M bicarbonate buffer, pH 9.5, for 16 h at 4°C. Following 3 washings with phosphatebuffered saline (PBS), pH 7.4, containing 0.05% Tween 20 (PBS-T20), the plates were blocked with 5 % bovine serum albumin (BSA) for 4 h. After washing, 100 pi of the diluted rat or human sera was added to the plates and incubated for 3 h. Following 3 washings, 125I-labelled goat antirat Ig (1 X 105 cpm) or goat anti human Ig (1 X 105 cpm) was incubated for 3 h at 4 °C. The plates were then washed 5 times with PBS-T20, the wells were cut and the radioactivity was counted in a gamma-counter. The assay was done in triplicate and the mean values were presented. Detection o f Anti-Id Antibodies Inhibition Assay. This assay was performed as de scribed [19], Fifty microliters of 125I-labelled MoAb 17-1A (1 X 105 cpm) was incubated for 2 h at 4 °C with 50 pi of diluted rat serum. Then, the mixture was added to 17-lA-bearing colonic carcinoma SW1116 cells (1 X lOVwell). At the end of another 2 h of incu bation at 4 ° C, the cells were washed 5 times with PBS, and the bound radioactivity was counted in a gammacounter. Results were expressed as the percentage of binding inhibition and were compared with the con trol serum which had been immunized with the irrele vant MoAb or with preimmune serum.
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ministered MoAb 17-1A as another mecha nism for an antitumor effect [4, 10, 11], Inves tigations have been undertaken to ascertain whether the idiotype network system has an important role to play in the regulation of immune responses [ 12, 13], as such responses may eventually result in the destruction of tumor cells. Therefore, it is important to mea sure the levels of anti-id antibodies as well as antimouse immunoglobulin (Ig) antibodies and to analyze the mechanism of possible immunotherapeutic effects on these patients. However, there have been some difficulties in detecting anti-id antibodies in the sera of patients who have been injected with murine MoAb, since antimouse Ig antibodies, as well as anti-id antibodies, may simultaneously be induced by and affect the assays. Herlyn et al. [ 10] have presented a specific assay system for the detection of anti-id anti bodies in patient sera. However, the proce dure to prepare the purified goat anti-id anti bodies for the assay requires many steps. In the present study, we established a simpler assay for the detection of anti-id anti bodies in a xenogeneic model system and investigated the incidence and kinetics of in duction of anti-id antibodies and antimouse Ig antibodies in cancer patients administered murine MoAb 17-1 A.
Clinical Studies Serum from 13 metastatic or recurrent cancer (8 gastric and 5 colonic cancer) patients was used in this study. All patients were administered purified murine MoAb 17-1A in the phase I trial in Japan (Chairman: Prof. T. Taguchi, Osaka University School of Medi cine). These patients were injected systemically with one dose of MoAb (25, 100 or 1,000 mg) in a single infusion. The patient sera for detection of antimouse Ig or anti-ld antibodies were obtained at 1-week inter vals after the administration of MoAb 17-1 A. The sandwich assay and the modified sandwich assay de scribed above were used in order to detect antimouse Ig and anti-id antibodies, respectively.
Results
Detection o f Antimouse Ig Antibodies in Rat Serum Elicited with Murine MoAbs In order to detect antimouse Ig antibodies in a xenogeneic system, as a model for human patients who were injected with murine MoAb, rats were immunized with murine MoAbs. Serially diluted sera from rats immu nized with purified murine MoAb 17-1A (IgG2a) or irrelevant murine MoAb M7-049 (IgG2a) were tested in the binding assay with the use of coated MoAb 17-1A as a catcher and 125I-labelled goat antirat Ig antibodies as a tracer. As shown in figure 1, not only the rat sera immunized with MoAb 17-1A but also those immunized with MoAb M7-049 reacted
228
remarkably with MoAb 17-1 A, whereas preimmune rat sera did not. Detection o f Anti-Id Antibodies in Immunized Rat Sera Two different types of assay were estab lished to detect anti-id antibodies specific to MoAb 17-1A in this xenogeneic system. The first was the inhibition assay, which demonstrates the inhibitory activity of rat sera against the binding of l25I-labelled MoAb 17-1A to antigen. Human colonic carcinoma SW1116 cells, which express 17-1A antigen, were used as targets in this assay. The serum of rats who had been immunized with MoAb 17-1A remarkably inhibited the binding of l25I-labelled MoAb 17-1A to the cells, as shown in figure 2, while the serum of rats who had been immunized with MoAb M7-049 failed to inhibit. This inhibition, however, was affected when MoAb 17-1A was added to the serum (data not shown). More than 10 pg/ ml of MoAb 17-1A in the serum blocked the binding inhibition, suggesting that this assay should not be utilized when a concentration of MoAb 17-1A is higher than 10 pg/ml in the serum. The second system to detect anti-id anti bodies is the sandwich assay with use of MoAb 17-1 A. Serum of rats who had been immunized with murine MoAb 17-1A was applied to this assay. The binding activities of rat anti-MoAb 17-1A and anti-MoAb M7-049 seru m to MoAb 17-1A were detected in a con centration-dependent manner, while that of preimmune rat serum was not, as shown in figure 3a. This reaction can be explained as follows: the antimouse Ig antibodies in antiMoAb 17-1A serum and also in anti-MoAb M7-049 serum, which were induced simulta neously in rats, reacted with mouse MoAb 171A, which was used as a catcher and a tracer. There is a possibility, however, that the antiid antibodies to MoAb 17-1A were induced.
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Sandwich Assay. As described elsewhere [18], a sandwich assay was performed. Briefly, MoAb-17-lAcoated microtiter plates were blocked with 5 % BSA for 4 h, and then 100 pi of diluted rat serum was added and incubated for 4 h. Following 3 washings, the plates were incubated with l25l-labclled MoAb I7-1A for4 h. The plates were then washed 5 times and the radioac tivity was counted. On the other hand, a modified sandw'ich assay was also done, utilizing 50 pi of 1:10-diluted normal mouse serum which was preincubated with 50 pi of diluted rat or human serum before being applied to MoAb-17-1Acoatcd plates. Both assays were done in triplicate, and the mean values were presented.
1:40
1:160 1:80
1:320
1:640 1:2,560 1:10.240 1:1,280 1:5,120
Serial dilution of rat sera
Dilution of rat sera
Fig. 1. Detection of antimouse Ig antibodies against murine MoAb 17-1A in sera of rats immunized with murine MoAb 17-1A. Rat anti-MoAb 17-1A serum (o), anti-MoAb M7-049 serum (A ) and preimmune serum (□) were serially diluted with PBS and tested, utilizing coated MoAb 17-1A as a catcher and l25l-labelled antirat Ig antibodies as a tracer. Fig. 2. Inhibitory activities of diluted rat antiMoAb 17-1A serum (o), anti-MoAb M7-049 serum (A ) and preimmune serum (□) on l25I-labelled MoAb 17-1A binding to colonic carcinoma SW 1116 cells.
Fig. 3. Sandwich assay for the detection of anti-id antibodies to MoAb 17-1 A. a Serially diluted rat antiMoAb 17-1A serum (o), anti-MoAb M7-049 serum ( a ) and preimmune serum (□) were incubated with MoAb-17-IA-coated microtiter plates and then tested for reactivity of l25l -labelled MoAb 17-1 A. b The mix ture of serially diluted rat anti-MoAb 17-1A serum (o), anti-MoAb M7-049 serum (A ) or preimmune serum (□), and 1:10-diluted normal mouse serum was tested in a modified sandwich assay.
In order to reduce the effect of coinduced antimouse Ig (non-anti-Id) antibodies in the rat, the excess normal mouse serum was incu bated with rat sera before application of these sera to the sandwich assay. Figure 3b shows the results of the sandwich assay using the mixture of MoAb immunizing rat sera and
normal mouse sera to neutralize the activity of the anti mouse Ig antibodies. The reactivity of MoAb M7-049 immuniz ing rat serum was completely reduced by the addition of normal mouse serum, although that of MoAb 17-1A immunizing rat serum retained the reactivity. The relative percent
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3a
age of antimouse activity which was due to anti-id would be approximately 60% for MoAb 17-1A and 9% for MoAb M7-049. A sandwich assay using MoAb M7-049 as a catcher and a tracer was also performed to measure the anti-ld antibodies to MoAb M7049. It showed a similar tendency (fig. 4a.b) to that of MoAb 17-1 A, suggesting that this modified sandwich assay is useful for detect ing anti-id antibodies. Anti-constant region antibodies in rat serum have been mainly responsible for disturbing sandwich assays to detect anti-id antibodies. In order to deter mine the dose (or volume) of normal mouse serum to be preincubated with rat sera, the neutralizing activities of normal mouse se rum were tested in a sandwich assay. As shown in figure 5, a 1:50 dilution of normal mouse serum was sufficient to neutralize the antimouse Ig antibodies which were induced in 1:10-diluted rat sera. Addition of normal mouse serum even reduced the binding of 17-1A up to approximately 15% (26,000 cpm, at 1:5.000 dilution, to 22,000 cpm, at 1:50 dilution, bound). The presence of MoAb 17-1A in the rat serum is unlikely to affect this sandwich assay, since the concen tration of MoAb 17-1A in the serum should be too low.
Fig. 4. Sandwich assay for detection of anti-ld anti bodies to MoAb M7-049. Immunoassay was per formed as described in figure 3 using MoAb M7-049 as the capture and detection antibody, a, b Rat antiMoAb 17-1A serum (o), anti-MoAb M7-049 scrum (A) and preimmune serum (□).
Kinetics o f the Anti-Id Antibodies and Antimouse Ig Antibodies Induced with MoAb 17-1A The rats immunized with KLH-conjugated MoAb 17-1A were bled on days 7, 21, 42, 49 and 60 after the immunization, and the ob tained sera were used to measure both anti-id antibodies and antimouse Ig antibodies. The anti-ld antibodies to MoAb 17-1A were grad ually induced in rats and continued to be gen erated even after 60 days (fig. 6). On the other hand, the antimouse Ig antibodies were rap idly induced after the immunization and kept a high titer.
Detection o f Anti-Id Antibodies and Antimouse Antibodies in Patient Sera Thirteen patients with gastrointestinal ad enocarcinoma were injected with a purified sterile pyrogen-free preparation of MoAb 171A by a single infusion. Four of the 13 pa tients received 1,000 mg, 4 patients 100 mg and 5 patients 25 mg of MoAb 17-1 A. As shown in figure 7, 7 of the 13 patients who received the MoAb injection developed anti-ld antibodies to MoAb 17-1 A; 5 patients (cases 3, 4, 5, 11 and 12) showed a peak level
Dilution of rat sera
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Dilution of rat sera
Fig. 5. The effect of normal mouse serum on neu tralizing the rat antimouse Ig antibodies in a modified sandwich assay. Serially diluted normal mouse sera were preincubated with 1:10-diluted rat anti-MoAb 17-1A serum (o), anti-MoAb M7-049 serum (A) and preimmunc scrum (□).
Fig. 6. Changes in the levels of anti-id antibodies (o) and antimouse Ig antibodies (•) in the serum of rats
immunized with MoAb 17-1 A. Rat sera (1:10 and 1:100 dilution) were used to detect anti-id antibodies in a modified sandwich assay and antimouse Ig anti bodies in a binding assay (coated MoAb 17-1A and l25I-label!ed antirat Ig antibodies).
Table 1. Induction of anti-MoAb 17-1A antibodies and anti-id antibodies in the sera of patients by adminis tration of murine MoAb 17-1A
Induction of antibodies in patients
MoAb 17-1A injected 1,000 mg
100 mg
25 mg
Anti-MoAb 17-1A antibodies
4/4
100%
4/4
100%
4/5
80%
12/13
92%
Anti-Id antibodies
2/4
50%
3/4
75%
2/5
40%
7/13
54%
1A antibodies including both anti-constant region and anti-id antibodies, and their levels in sera changed in a pattern similar to those of anti-id antibodies. The highest level in the above 5 patients (cases 3, 4, 5, 11 and 12) was detected 2-3 weeks after the injection, and the level had no direct relation to the dose of MoAb 17-1A (fig. 7). The summary of the induction of antibodies in patients is shown in table 1.
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of anti-id antibodies 2-3 weeks following the administration of MoAb, whereas the other 2 patients (cases 6 and 8) at 6-8 weeks. Both cases 9 and 13 appear to have developed negligible antimouse Ig and anti-id. The induction levels of anti-id antibodies were unrelated to the administered doses of MoAb 17-1 A. With regard to human anti mouse Ig antibodies in patient sera, 12 of the 13 patients developed human anti-MoAb 17-
total
30.000
4,000
Case 1
20.000
30,000
Case 1
3,000
10,000 •
4,000
Case 5
20,000 | "
2,000
Case 5
3,000
P /v v .
10,000
2,000 /* •• •
0
1,000 0
4
8
12
0
4
8
12
0
4
8
12
1,000
0 0
4
8
12
0
4
8
12
a
b
232
modified sandwich assay were used for the detection of respective antibodies, a 4 cases injected with 1,000 mg MoAb. b 4 cases injected with 100 mg MoAb. c 5 cases injected with 25 mg MoAb.
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Fig. 7. Detection of human anti-MoAb 17-1A anti bodies (•. left panels) and anti-ld antibodies (o, right panels) in the sera of 13 patients who received murine MoAb 17-1A by a single infusion. Sandwich assay and
30.000
cpm
20.000 10,000
0 0
4
8
12
0
4
8
12
0
4
8
12
0
4
8
12
30.000
cpm
20.000 10,000
.....
cpm
cpm
0
30.000
4.000
Case 13
20.000
3.000
10.000 . \
2.000
Case 13
«
w*
1.000 4
8
Weeks
c
12
D
4
8
Weeks
12
Although several assays to detect anti-id antibodies generated in a xenogeneic system have been tried, they have not been feasible for a routine test since the specificity of the assays was not satisfactory and/or the assays were complicated. Herlyn et al. [10] reported on assays to detect anti-id antibodies reacting with combining and non-combining site-re lated idiotypes in murine MoAb 17-1 A. In that assay, it was necessary to prepare poly clonal anti-id antibodies to MoAb 17-1A which were isolated from animal sera (rabbit or goat) following absorption of the sera with the use of immunoabsorbent columns which were prepared with idiotypically unrelated MoAb of the same isotype (IgG2a). We tried to apply two different types of assays, one an inhibition assay and the other a sandwich assay, for the detection of xeno geneic anti-id antibodies. The inhibition as say demonstrates the inhibitory activity of rat serum generated with murine MoAb 17-1A on the binding o f 125I-labelled MoAb 17-1A to antigen-bearing cells. As shown in figure 2, MoAb 17-1A immunizing rat serum inhibited 125l-labcllcd MoAb 17-1A binding to the cells, whereas neither preimmune rat serum nor MoAb M7-049 immunizing rat serum were inhibitory. These results suggest that the in hibitory activity is not affected by antimouse Ig antibodies. The sandwich assay, which was established to detect anti-id antibodies in a syngeneic sys tem as described elsewhere [ 18], was applied to detect xenogeneic anti-ld antibodies. Both rat anti-MoAb 17-1A and anti-MoAb M7-049 sera reacted remarkably with murine MoAb 17-1A in the unmodified sandwich assay (fig. 3a). suggesting that not only anti-id anti bodies specific to MoAb 17-1A but also anti mouse Ig antibodies were detected in this assay.
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Discussion
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ually decreased (data not shown). These data are similar to those reported by Herlvn et al. [Hi-
Immune responses in cancer patients fol lowing a single infusion of MoAb 17-1A were investigated. As a result, the induction level of anti-id and antimouse Ig antibodies and dura tion time were found to be unrelated to the injection doses (25, 100 or 1,000 mg) of MoAb 17-1A (fig. 7a-c, table 1). Fifty-four and 92% of the patients developed anti-id and antimouse Ig antibodies, respectively. The incidence of positivity for induction of anti-id antibodies is almost the same as that in the phase II study reported by Sears et al. [20], The levels of anti-Id and antimouse Ig antibodies simultaneously fluctuated and their levels in most of the cases maximized 23 weeks after the injection. A pharmacoki netic study by Khazaeli et al. [3] has shown that most patients had an antibody response with peak levels occurring 15-20 days after infusion in the phase I trial. With regard to the differences between anti-Id antibody in duction noted in rat sera and that of patient sera, it is speculated that rats immunized with intraperitoneal injection of KLH-conjugated MoAb in Freund’s adjuvant had a continuous induction of anti-Id antibodies, while patients 661 blistered a single infusion of MoAb showed a rapid increase and reduction in antiId antibodies in the sera. Hcrlyn et al. [ 11 ] have indicated that a sin gle injection of MoAb to a cancer patient could continue inducing anti-Id antibodies (but not antimouse Ig antibodies) for 400 days after administration. Our studies [21, 22] have also suggested that syngeneic anti-Id an tibodies could be detected for more than 200 days after intraperitoneal injection of KLHconjugated MoAb. The reason why contin uous production of anti-Id antibodies was maintained was unclear at the time of this study. This phenomenon, however, may be
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The addition of normal mouse sera to im munized rat sera and their preincubation are of use in order to eliminate the influence of rat antimouse Ig antibodies. As shown in fig ure 3b, rat anti-id antibodies to murine MoAb 17-1A could be specifically detected in the modified sandwich assay, using the excess of normal mouse serum to neutralize antimouse Ig antibody activities. When rat anti-MoAb M7-049 serum was tested, antiid antibodies specific to MoAb M7-049 were detected in the modified sandwich assay us ing MoAb M 7-049 (fig. 4b) but were not spe cifically detected in the unmodified proce dure (fig. 4a). These results suggest that the modified sandwich assay is of use to detect anti-id antibodies specific to the immunizing MoAb. Furthermore, it is suggested that it was necessary to add a 1:50 dilution of normal mouse serum to rat sera diluted 1:10 in order to reduce the effects of anti mouse Ig and other antibodies (fig. 5). Normal mouse sera showed more effective neutralizing ability than an unrelated murine MoAb (IgG2a; data not shown), since they contain a panel of murine antibodies with different isotypes and different amino acid sequences in the frame work of the variable regions. Using the modified sandwich assay to de tect anti-id antibodies and the binding assay with coated MoAb 17-1A and l25I-labelled antirat Igs to detect antimouse Ig antibodies, the time course of anti-id and antimouse Ig antibody production was investigated in rats which were immunized 3 times with murine MoAb 17-1A at 0, 7 and 14 days. Figure 6 shows that the anti-id antibodies were gener ated gradually and continued to be produced for up to 60 days and that antimouse anti bodies were rapidly induced after the first infusion of MoAb. The high level of anti-id antibodies in rat sera was maintained after 60 days, while the anti mouse antibodies grad
important in the hosts’ immune response against tumor progression. Although possible correlations between the clinical status of the patients and their im mune response to the mouse monoclonal anti body were not clear at the time of this study, they should be analyzed in the near future. From the viewpoint of the idiotype network theory, internal-image-bcaring anti-id anti bodies may be of use for immunotherapy to induce an antitumor immune response as an antigen-specific immunomodulator. Based on this strategy, we have developed and analyzed the internal-image-bearing anti-id MoAbs which are related to carcinocmbryonic anti gen [14], Further studies are now under way in our laboratory to clarify the relationship between the induction of anti-id antibodies
and the prognosis of cancer patients and the contribution of cellular immunity as well as humoral immunity.
Acknowledgements This work was supported by a Grant-in-Aid for Co operative Research (AY, KI), by a Grant-in-Aid for Scientific Research on Priority Areas (KI) and by a Grant-in-Aid for Cancer Research (MT, KI, AY) from the Ministry of Education, Science and Culture, and was also supported by Grants for Cancer Research (AY) from the Ministry of Health and Welfare, Japan. The technical assistance of Ms. M. Nosaka, Ms. K. Kawamata and Ms. M. Ohe is greatly appreciated. We especially thank Dr. Y. Hinoda and Dr. T. Sugiyama for helpful advice, Mr. T. Mimura for grateful support and Ms. H. Yamashita for secretarial assistance.
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